Large serine recombinases and methods of use

Abstract

Disclosed herein are composition and methods of modifying a target double-stranded DNA (dsDNA) molecule in a cell. Systems, compositions, and methods may comprise a recombinase (e.g., a large serine recombinase (LSR)), a CRISPR-associated (Cas) protein, an RNA-dependent DNA polymerase, and / or one or more guide nucleic acids, or uses thereof.

Description

LARGE SERINE RECOMBINASES AND METHODS OF USECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application 63 / 503,327, filed May 19, 202.3; U.S. Provisional Application 63 / 582,344, filed September 13. 2023; and U.S. Provisional Application 63 / 606.194, filed December 5, 2023, each of which are herein incorporated by reference in their entireties.REFERENCE TO THE ELECTRONIC SEQUENCE FILE100021 The contents of the electronic sequence listing (MAB1 037 ()3W() SeqList ST26.xml; Size: 10,944,919 bytes; and Date of Creation: May 17, 2024) are herein incorporated by reference in its entirety'.BACKGROUND

[0003] Large serine recombinases (LSRs), also sometimes referred to as integrases, catalyze the movement of DNA elements into and out of bacterial chromosomes using site-specific recombination between short DNA "attachment sites". The LSRs that function as bacteriophage integrases cany' out integration between attachment sites in the phage (attP) and in the host (attB).

[0004] Site-specific recombinases, such as large serine recombinases, have evolved to catalyze the transfer of large genetic payloads that are often tens of kilobases in length, from one organism to another, without relying on recipient genetic repair machinery. By recognizing attachment sites found m donor and target sequence DNA, recombinases are capable of catalyzing target cleavage, strand exchange and DNA rejoining within their synaptic complexes. This mechanism enables site-specific DNA insertion without requiring any cellular cofactors and without generating exposed DSBs. Furthermore, unlike tyrosine recombinases, LSRs do not have to be engineered to prevent excision of the integration product once integration occurs. An LSR is usually 400-700 amino acids in length including a catalytic site and several domains that are used for sequence recognition and placement for the integration to occur.

[0005] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated proteins (Cas proteins), sometimes referred to as a CRISPR / Cas system, were first identified in certain bacterial species and are now understood to form part of a prokaryotic acquired immune system. CRISPR / Cas systems provide immunity in bacteria and archaea againstviruses and plasmids by targeting the nucleic acids of the viruses and plasmids in a sequencespecific manner Native systems contain a CRISPR array, which includes direct repeats flanking short spacer sequences that, in part, guide Cas proteins to their targets. The discovery of CRISPR / Cas systems has revolutionized the field of genomic manipulation and engineering, and therapeutic applications of these systems are being explored.SUMMARY

[0006] Disclosed herein are novel compositions of large serine recombinases (LSRs) and uses thereof

[0007] In some aspects, the disclosure provides systems and methods for using CRISPR systems to generate attachment sites where integration of a donor DNA via an LSR is desired.

[0008] In some embodiments, the present disclosure provides a system for the modification of a target nucleic acid comprising: a recombinase or a nucleic acid encoding a recombinase; and a donor DNA vector comprising a first recombinase recognition sequence (RRS-1 ). wherein the recombinase comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 1-3918 and 9761-9763, and wherein the target nucleic acid comprises a second RRS (RRS-2).

[0009] In some embodiments, the recombinase comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NOs: 1-2523 and 9761 -9763, the RRS-1 comprises a nucleotide sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs:6442-8964, 9740-9760, and 9764-9791, and the RRS-2 comprises a nucleotide sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 3919-6441, 9719-9739, and 9792-9819, wherem the recombinase, RRS-1 and RRS-2 are in the same row of TABLE 1 or TABLE 2

[0010] In some embodiments, the RRS-l and / or RRS-2 is located in or near a safe harbor locus In some embodiments, the safe harbor locus is near an AAPS1 (PPPIR12C) gene, an ALB gene, an ANGPTL3 gene, an APOC3 gene, an ASGR2 gene, a CCR5 gene, a FIX (F9) gene, a G6PC gene, a GYS2 gene, an HGD gene, a LP(A) gene, a PCSK9 gene, a SERF INA I gene, a TF gene, and a TTR gene, and an intron thereof. In some embodiments, the RRS-1 and / or RRS-2 is located in or near a target sequence of the human albumin gene (ALB) or AA VS1 gene.

[0011] In some embodiments, the target sequence is selected from a nucleotide sequence that is at least 80%, at least 90%. at least 95% or 100% identical to a sequence selected from SEQ ID NGs: 8965-9266, and wherein the target sequence is in the same row of TABLE 1 as the recombinase. RRS-1, and RRS-2.

[0012] In some embodiments, the donor DNA vector is a circularized DNA molecule.

[0013] In some embodiments, the recombinase or nucleic acid encoding the recombinase, and the donor DNA vector are combined in a single composition. In some embodiments, the nucleic acid encoding the recombinase comprises the donor DNA.

[0014] In some embodiments, the present disclosure provides methods of modifying a target nucleic acid comprising contacting the target nucleic acid with the components of a composition or system described herein.

[0015] In some embodiments, the present disclosure provides methods of modifying a target nucleic acid in a cell, the method comprising contacting the cell with the components of a composition or system described herein

[0016] In some embodiments, the present disclosure provides compositions for the modification of a target nucleic acid, wherein the compositions comprise a fusion protein or a nucleic acid encoding the fusion protein, wherein the fusion protein comprises: a recombinase comprises an ammo acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%. at least 95%. at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 1 - 3918 and 9761-9763; an effector protein; and an RNA dependent DNA polymerase (RDDP).

[0017] In some embodiments, the effector protein is a CRISPR associated (C ’as) protein. In some embodiments, the Cas protein is a Type V Cas protein. In some embodiments, the Cas protein comprises an amino acid sequence that is at least 75%, at least 80%. at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs. 9267-9401 and 9698-9701 . In some embodiments, the length of the Cas protein is 350 to 550 amino acids. In some embodiments, the length of the Cas protein is 550 to 800 amino acids.In some embodiments, the Cas protein comprises nickase activity.

[0018] In some embodiments, the RDDP comprises an ammo acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 9402-9482.

[0019] In some embodiments, the compositions comprise an extended guide nucleic acid, wherein the extended guide nucleic acid comprises: i) a protein binding sequence, wherein the effector protein is capable of binding the protein binding sequence, ii) a spacer sequence thathybridises to a first target sequence on a first strand of the target nucleic acid, iii) a primer binding sequence, optionally wherein a portion of the primer binding sequence hybridizes to a first portion of the second strand of the target nucleic acid; and iv) a template sequence that hybridizes to a second portion of the second strand of the target nucleic acid.

[0020] In some embodiments, the composition comprises a first guide nucleic acid, wherein the extended guide nucleic acid comprises: i) a protein binding sequence, wherein the effector protein is capable of binding the protein binding sequence, ii) a spacer sequence that hybridizes to a first target sequence on a first strand of the target nucleic acid, iii) a primer binding sequence, optionally wherein a portion of the primer binding sequence hybridizes to a first portion of the first strand of the target nucleic acid; and iv) a template sequence that hybridizes to a second portion of the first strand of the target nucleic acid.

[0021] In some embodiments, compositions comprise a donor DNA vector, wherein the donor DNA vector first RRS (RRS-1). In some embodiments, the template sequence of the extended guide nucleic acid comprises at least a portion of a first or second RRS, and the target sequence optionally cotriprises a portion of the first or second RRS. In some embodiments, the composition comprises a second guide nucleic acid, wherein the guide nucleic acid comprises a protein binding sequence and a spacer sequence that hybridizes to a second target sequence on the second strand of the target nucleic acid.

[0022] In some embodiments, the fusion protein comprises a nuclear localization signal.

[0023] In some embodiments, the present disclosure provides methods of modifying a target nucleic acid comprising contacting the target nucleic acid with a composition provided herein. In some embodiments, the present disclosure provides methods of modifying a target nucleic acid in a cell, the method comprising contacting the cell with the components of a system of described herein.

[0024] In some aspects, disclosed herein are systems for the modification of a target nucleic acid comprising: (a) a recombinase or a nucleic acid encoding a recombinase; (b) a donor DNA vector comprising a first recombinase recognition sequence (RRS-1). wherein the target nucleic acid comprises a second RRS (RRS-2); and wherein(a) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%. or 100% identical to SEQ ID NO: 3; the RRS-1 comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 6444 or 9766;and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 3921 or 9794;(b) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 16; the R.RS-1 comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%. at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 6457, 9740, or 9764; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 3934, 9719, or 9792;(c) the recombinase comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 18; the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 6459, 9743, or 9767: and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%. at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 3936, 9722, or 9795;(d) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 20; the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 6461 , 9741 , or 9765; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 3938. 9720, or 9793;(e) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%. at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 350; the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 6791, 9756, or9780; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 4268, 9735, or 9808;(f) the recombinase comprises or consists of an amino acid sequence that is at least 70%, al least 75%, at least 80%, at least 85%, at least 90%, at least 95%, al least 98%, at least 99%. or 100% identical to SEQ ID NO: 441; the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%. at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 6882, 9757, 9781, or 9784 ; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%. at least 75%. at least 80%. at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 4359, 9736. 9809, or 9812;(g) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 454; the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 6895, 9751 , or 9775; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%. at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 4372, 9730, or 9803;(h) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 455; the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 6896, 9750, or 9774; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEC) ID NOs: 4373. 9729, or 9802;(i) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 569; the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 7010, 9748, or911T. and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 4487, 9727, or 9800;(f) the recombinase comprises or consists of an amino acid sequence that is at least 70%, al least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 591: the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%. at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 7032, 9755, or 9779; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 4509, 9734, or 9807;(k) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 020: the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 7061 , 9758, or 9782: and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%. at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 4538, 9737, or 9810;(l) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 955: the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 7396 or 9791 ; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to any one of SEQ ID NOs: 4873 or 9819;(m) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%. at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1 105; the RRS-1 comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 7546, 9760, or9783; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5023, 9739, or 9811;(n) the recombinase comprises or consists of an amino acid sequence that is at ieast 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 1112; the RRS-l comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 7553, 9754, or 9778; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5030, 9733, or 9806;(o) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1162; the RRS-l comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%. at least 99%. or 100% identical to any one of SEQ ID NOs: 7603 or 9789; and the RRS-2 comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5080 or 9817;(p) the recombinase comprises or consists of an amino acid sequence that is at ieast 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 1319; the RRS-l comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 7760, 9745, or 9769; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at ieast 85%, at least 90%, at ieast 95%, at ieast 98%, at ieast 99%, or 100% identical to any one of SEQ ID NOs: 5237. 9724, or 9797;(q) the recombinase comprises or consists of an amino acid sequence that is al least 70%, at least 75%, at least 80%. at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1452; the RRS-l comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 7893, 9749, or9773; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5370, 9728, or 9801;(r) the recombinase comprises or consists of an amino acid sequence that is at least 70%, al least 75%, at least 80%, at least 85%, at least 90%, at least 95%, al least 98%, at least 99%. or 100% identical to SEQ ID NO: 1518; the RRS-l comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at feast 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 7959, 9752, or 9776; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5436, 973 I , or 9804;(s) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1735; the RRS-l comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%. at least 99%. or 100% identical to any one of SEQ ID NOs: 8176 or 9788; and the RRS-2 comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to any one of SEQ ID NOs: 5653 or 9816;(t) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 1944; the RRS-l comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 8385, 9746, or 9770; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5862. 9725, or 9798;(u) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%. at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1966; the RRS-l comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 8407, 9753, or9777; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5884, 9732, or 9805;(v) the recombinase comprises or consists of an amino acid sequence that is at least 70%, al least 75%, at least 80%, at least 85%, at least 90%, at least 95%, al least 98%, at least 99%. or 100% identical to SEQ ID NO: 2050; the RRS-l comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at feast 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 8491, 9744, or 9768; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5968, 9723, or 9796;(w) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2068; the RRS-l comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%. at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 8509, 9759, or 9790; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%. at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5986, 9738, or 9818;(x) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 2172; the RRS-l comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 8613, 9747, or 9771; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 6090. 9726, or 9799;(y) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%. at least 85%>, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9761 ; the RRS-l comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9785; and the RRS-2.comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9813;(z) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 9762: the RRS-l comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9786; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%. at least 75%, at least 80%, at least 85%. at least 90%. at least 95%. at least 98%. at least 99%. or 100% identical to SEQ ID NO: 9814: or(aa) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9763; the RRS-l comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9787; and the RRS-2 comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9815.{0025] A system for the modification of a target nucleic acid comprising (a) a recombinase or a nucleic acid encoding a recombinase; (b) a donor DNA vector comprising a first recombinase recognition sequence (RRS-l), wherein the target nucleic acid comprises a second RRS (RRS-2), wherein the recombinase comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a protein any one of SEQ ID of column 3 of TABLE 16, and (c) wherein at least one of the RRS- 1 and RRS-2 comprises a corresponding kmer sequence of TABLE 16; wherein in the protein and the kmer are on the same line of TABLE 16,DESCRIPTION OF FIGURES

[0026] FIG, 1 A - FIG. IC illustrate examples of DNA rearrangement mediated by large serine recombinases and recombination directionality factors (RDFs). FIG. 1A shows DNA integration mediated by RRS-lKRRS-2 + LSR recombination. FIG. IB shows recombinationI Ibetween / ?7?A sites in linear DNA. FIG. 1C shows DNA inversion through RRS-1 -< RRS-2 recombination.

[0027] FIG. 2A - FIG. 2C illustrate exemplary methods on modifying a target nucleic acid sequence with the large serine recombinase systems described herein. FIG. 2A illustrates methods utilizing one pair of RRS-1 x RRS-2 sites. FIG. 2B illustrates methods utilizing two pairs of RRS-1 x RRS-2 sites and one LSR. FIG. 2C illustrates methods utilizing two pairs of RRS-1 x RRS-2 sites and two LSRs. FIG. 2A - FIG. 2C illustrates exemplary methods of target nucleic acid modification, wherein the donor nucleic acid is present as a circular RNA.Similar methods are contemplated, wherein the donor nucleic acid is present as a liner nucleic acid.

[0028] FIG. 3 shows an exemplary gene editing system comprising an extended guide RNA with the primer binding sequence and template sequence located 5' of a repeat sequence and a spacer sequence. The effector protein is fused to the RNA-directed DNA polymerase (RDDP). System components shown in FIG. 3 are exemplary and non-limiting. For instance, the 5’ extended rtgRNA may comprise additional nucleotides beyond those labeled as spacer, repeat, linker, PBS and RT template.

[0029] FIG. 4 shows an exemplary gene editing system comprising an extended guide RNA with the primer binding sequence and template sequence located 3’ of a repeat sequence and a spacer sequence. 'The effector protein is fused to the RNA-directed DNA polymerase (RDDP), System components shown in FIG. 4 are exemplar} and non-limiting. For instance, the 5’ extended rtgRNA may comprise additional nucleotides beyond those labeled as spacer, repeat, linker, PBS and RT template.

[0030] FIGs. 5A-5B show exemplary split protein RNA design gene editing systems comprising a guide RN A with the repeat and spacer sequences separate from the primer binding sequence and template sequence. The effector protein is separate from the RDDP The effector protein is localized to the target nucleic acid via the guide RNA where it can nick the target nucleic acid. The primer biding sequence and template sequence are linked to an MS2 aptamer. The RDDP is fused to an MS2 coat protein that is capable of binding the MS2 aptamer, thereby localizing the RDDP to the nicked DNA. System components shown in FIGs. 5A and 5B are exemplary and non-limiting. For instance, the gRNA and / or retRNA may comprise additional nucleotides beyond those labeled as spacer, repeat, linker, PBS and RT template.

[0033] FIG. 6 shows an overview of a system comprising CasM.265466 for precise editing.CasM.265466 generates a double-stranded break on target DNA. A retRNA hybridizes withthe non-target strand (as shown) or target strand (not shown) via complementarity between the PBS region and the target DNA. An RDDP is recruited to the target site by binding of a fused MS2 coat protein to the MS2 aptamer of the retRNA. The RNA / DNA hybrid serves as a binding substrate for the RDDP. which then synthesizes new cDNA along the exposed 3" end of the target DNA using the RT template as a reverse transcription template. Edits encoded in the RT template are thereby written into the genome.

[0032] FIG. 7 shows incorporation of a donor DNA reporter (mCherry) into safe harbor loci (albumin and A4FS7) of mammalian cells by LSRs disclosed herein.

[0033] FIG. 8 shows a representative diagram of a promoter swap assay via DNA inversion. as also exemplified in FIG. 1C.

[0034] FIG, 9 shows a representative diagram of a reporter swap assay via DNA inversion, as also exemplified in FIG. IC.

[0035] FIG. 10 shows the results of a promoter swap assay demonstrating that the LSR proteins have recombinase activity in mammalian cells.

[0036] FIG. 11 show's the results of a reporter swap assay demonstrating that the LSR proteins have recombinase activity in mammalian cells.

[0037] FIG. 12 shows the results of a reporter swap assay demonstrating that the LSR proteins can integrate large payloads into a gene sequence.

[0038] FIG. 13A-FIG. 13D show' the results of additional LSR proteins used in the reporter swap assay. FIG. 13A and FIG. 13C show7the results of the reporter swap assay as a percent of mCherry expression, which demonstrates recombination efficiency. FIG. 13B and FIG. 13D show the percent GFP expression, which demonstrates the overall transfection efficiency.

[0039] FIG. 14A-FIG. 14B show a representative assay (FIG. 14A) to test for the integration efficiency of a donor plasmid comprising an atB or attP site and the results (FIG. 14B) of said assay.DETAS LED DESCRIPTION

[0040] It is to be understood that both the foregoing general description and the following detailed description are exemplary7, and explanatory7only, and are not restrictive of the disclosure.

[0041] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles.books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.Definitions[00421 Unless otherwise indicated, all technical terms used herein have the same meaning as commonly understood by one of' ordinary ski 11 in the art. Unless otherwise indicated or obvious from context, the following terms have the following meanings:

[0043] As used in this specification and the appended claims, the singular forms ‘'a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.

[0044] Any reference to "or” herein is intended to encompass "and / or” unless otherwise stated. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0045] Use of the term “including” as well as other forms, such as “includes” and “’included,” is not limiting.

[0046] As used herein, the term “comprise” and its grammatical equivalents specifies the presence of stated features, integers, steps, operations, elements, and / or components, but does not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0047] As used herein, the term “about” in reference to a number or range of numbers is understood to mean the stated number and numbers + / - 10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range

[0048] The terms “% identical,” “% identity,” and “percent identity,” or grammatical equivalents thereof, with reference to an ammo acid sequence or nucleotide sequence, refer to the percent of residues that are identical between respective positions of two sequences when the two sequences are aligned for maximum sequence identity. The % identity is calculated by dividing the total number of the aligned residues by the number of the residues that are identical between the respective positions of the at least two sequences and multiplying by 100. Generally, computer programs can be employed for such calculations. Illustrative programs that compare and align pairs of sequences, include ALIGN (Myers and Miller, Comput Appl Biosci. 1988 Mar,4(l): l 1-7), FASTA (Pearson and Lipman, Proc Natl Acad Sci U S A. 1988 Apr; 85(8): 2444-8; Pearson, Methods Enzymol. 1990; ! 83:63-98) and gapped BLAST (Altschul et al.. Nucleic Acids Res. 1997 Sep 1:25(171:3389-40), BLAST?. BLASTN. or GCG (Devereux et al., Nucleic Acids Res. 1984 Jan 11;12(1 Pt l):387-95). To determine sequence identity, sequences can be aligned using various convenient methods and computer programs(e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.), available over the world wide web at sites including ncbi.nlm.nili.gov / BLAST, ebi.ac.uk / Tools / msa / tcoffee / , ebi.ac.uk / Tools / nisa / muscle / , mafft.cbrc.jp / alieiiment / software / . See, e.g., Altschul et al. (1990). J. Mol. Bioi. 215:403-10. [0049 j 'lire terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural. or derivatized nucleotide bases.

[0050] "Binding" as used herein (e g. with reference to an RNA-bmding domain of a polypeptide, binding to a target nucleic acid, and the like) refers to a non-covalent interaction between macromolecules (e.g.. between a protein and a nucleic acid; between a CAS polypeptide / guide RNA complex and a target nucleic acid; and the like). While in a state of non-covalent interaction, the macromolecules are said to be "associated" or "interacting" or "binding" (e.g., when a molecule X is said to interact with a molecule Y, it is meant the molecule X binds to molecule Y in a non-covalent manner). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), but some portions of a binding interaction may be sequence-specific. Binding interactions are generally characterized by a dissociation constant (KD) of less than 10"6M, less than 10’7M, less than 1O‘SM, less than 10'9M, less than 10',yM, less than IO'11M, less than 10'12M, less than IO’13M, less than I O'14M. or less than I O'15M. "Affinity" refers to the strength of binding, increased binding affinity being correlated with a lower KD.

[0051] By "binding domain" it is meant a protein or nucleic acid domain that is able to bind non-covalently to another molecule. A binding domain can bind to, for example, a DNA molecule (a DNA-binding domain), an RNA molecule (an RNA-binding domain) and / or a protein molecule (a protein-binding domain). In the case of a protein having a protein- binding domain, it can in some cases bind to itself (to form honiodimers, homotrimers, etc.) and / or it can bind to one or more regions of a different protein or proteins.

[0052] The term, “cis cleavage.” as used herein, refers to cleavage (hydrolysis of a phosphodi ester bond) of a target nucleic acid by a complex of an effector protein and a guide nucleic acid, wherein at least a portion of the guide nucleic acid is hybridized to at least aportion of the target nucleic acid. Cleavage may occur wi thin or directly adjacent to the portion of the target nucleic acid that is hybridized to the portion of the guide nucleic acid.

[0053] The terms, “cleave," “cleaving," and “cleavage,’' as used herein, with reference to a nucleic acid molecule or nuclease activity of an effector protein, refer to the hydrolysis of a phosphodiester bond of a nucleic acid molecule that results in breakage of that bond. The result of this breakage can be a nick (hydrolysis of a single phosphodiester bond on one side of a double-stranded molecule), single strand break (hydroly sis of a single phosphodiester bond on a single-stranded molecule) or double strand break (hydrolysis of two phosphodiester bonds on both sides of a double-stranded molecule) depending upon whether the nucleic acid molecule is single-stranded (e g.. ssDNA or ssRNA) or double-stranded (e.g., dsDNA) and the type of nuclease activity being catalyzed by the effector protein.

[0054] 'The terms, “complementary'’ and “complementarity,” as used herein, with reference to a nucleic acid molecule or nucleotide sequence, refer to the characteristic of a polynucleotide having nucleotides that base pair with their Watson-Crick counterparts (C with G; or A with T) in a reference nucleic acid. For example, when eveiy nucleotide in a polynucleotide forms a base pair with a reference nucleic acid, that polynucleotide is said to be 100% complementary to the reference nucleic acid. In a double stranded DNA or RNA sequence, the upper (sense) strand sequence is in general, understood as going in the direction from its 5'- to 3'-end, and the complementary sequence is thus understood as the sequence of the lower (antisense) strand in the same direction as the upper strand. Following the same logic, the reverse sequence is understood as the sequence of the upper strand in the direction from its 3'- to its 5 '-end, while the ’reverse complement' sequence or the ’reverse complementary’ sequence is understood as the sequence of the lower strand in the direction of its 5'- to its 3'-end. Each nucleotide in a double stranded DNA or RNA molecule that is paired with its Watson-Crick counterpart called its complementary nucleotide.

[0055] 'The term, “conservative substitution” as used herein refers to the replacement of one amino acid for another such that the replacement takes place within a family of ammo acids that are related in their side chains. Conversely, the term “non-conservative substitution” as used herein refers to the replacement, of one ammo acid residue for another that does not have a related side chain. Genetically encoded amino acids can be divided into four families having related side chains: (1) acidic (negatively charged): Asp (D), Glu (G): (2) basic (positively charged): Lys (K), Arg (R), His (H); (3) non-polar (hydrophobic): Cys (C), Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Met (M), Tip (W), Gly (G), Tyr (Y), with non-polar also beingsubdivided into: (i ) strongly hydrophobic: Ala (A), Vai (V), Leu (L), He (I), Met (M), Phe (F); and (ii) moderately hydrophobic: Gly (G), Pro (P), Cys (C), Tyr (Y), Trp (W); and (4) uncharged polar: Asn (N), Gin (Q), Ser (S), Thr (T). Ammo acids may be related by aliphatic, side chains: Gly (G). Ala (A), Vai (V), Leu (L). lie (1), Ser (S). Thr (T), with Ser (S) and Thr (T) optionally being grouped separately as aliphatic-bydroxyl. Amino acids may be related by aromatic side chains: Phe (F). Tyr (¥), Trp (W). Ammo acids may be related by amide side chains: Asn (N), Glu (Q). Amino acids may be related by sulfur-containing side chains: Cys (C) and Met (M).

[0056] The term, “clustered regularly interspaced short palindromic repeats (CRISPR).” as used herein, refers to a segment of DNA found in the genomes of certain prokaryotic organisms, including some bacteria and archaea, that includes repeated short sequences of nucleotides interspersed at regular intervals between unique sequences of nucleotides derived from another organism.

[0057] The term, ‘‘donor nucleic acid,” as used herein, refers to a nucleic acid that is (designed or intended to be) incorporated into a target nucleic acid or target sequence.

[0058] The term. “% editing efficiency,” as used herein, refers to the percent of target nucleic acids in a sample or population of cells exhibiting an edited target nucleic acid. Editing efficiency may also be referred to as % editing level or % edited. There are multiple approaches to evaluate % editing efficiency, including, but not limited to, next generation sequence and real time PCR.

[0059] The term, ‘'target nucleic acid,” as used herein, refers to a nucleic acid that is selected as the nucleic acid for modification, binding, hybridization or any other activity of or interaction with a nucleic acid, protein, polypeptide, or peptide described herein. A target nucleic acid may comprise RNA, DNA, or a combination thereof. A target nucleic acid may be single-stranded (e.g., single-stranded RNA or single-stranded DNA) or double-stranded(e.g.. double-stranded DNA)

[0060] The term, “target sequence.” as used herein, when used in reference to a target nucleic acid, refers to a sequence of nucleotides found within a target nucleic acid. Such a sequence of nucleotides can, for example, hybridize to a respective length portion of a guide nucleic acid. Hybridization of the guide nucleic acid to the target sequence may bring an effector protein into contact with the target nucleic acid.

[0061] A nucleotide sequence that “encodes” a particular polypeptide or protein, is a nucleotide sequence that is transcribed into mRNA (in the case of DNA) and / or is translated (in the case of mRNA) into a polypeptide.

[0062] The term “transgene” as used herein refers to a nucleotide sequence that is inserted into a cell for expression of said nucleotide sequence in the cell . A transgene is meant to include (1 ) a nucleotide sequence that is not naturally found in the cell (e.g., a heterologous nucleotide sequence); (2) a nucleotide sequence that is a mutant form of a nucleotide sequence naturally found in the cell into which it has been introduced; (3) a nucleoli de sequence that serves to add additional copies of the same (e.g.. exogenous or homologous) or a similar nucleotide sequence naturally occurring in the cell into which it has been introduced; or (4) a silent naturally occurring or homologous nucleotide sequence whose expression is induced in the cell into which it has been introduced A donor nucleic acid can comprise a transgene. The cell in which transgene expression occurs can be a target cell, such as a host cell.

[0063] The term, ‘"functional fragment,” as used herein, refers to a fragment of a protein that retains some function relative to the entire protein. Non-limiting examples of functions are nucleic acid binding, protein binding, nuclease activity, nickase activity, deaminase activity, demethylase activity, or acetylation activity. A functional fragment may be a recognized functional domain, e.g., a catalytic domain such as, but not limited to, a RuvC domain.

[0064] 'The terms, “fusion effector protein,” “fusion protein,” and “fusion polypeptide,” may be used interchangeably herein and refer to a protein comprising at least two heterologous polypeptides. Often a fusion effector protein comprises an effector protein and a fusion partner protein In general, the fusion partner protein is not an effector protein. Examples of fusion partner proteins are provided herein.

[0065] The terms “fusion partner protein” or "‘fusion partner,” as used herein, refer to a protein, polypeptide or peptide that is fused, or linked via a linker, to an effector protein. The fusion partner generally imparts some function to the fusion protein that is not provided by the effector protein.

[0066] The term, “effector protein,” as used herein, refers to a polypeptide that non-covalently binds to a guide nucleic acid to form a complex that contacts a target nucleic acid, wherein at least a portion of the guide nucleic acid hybridizes to a target sequence of the target nucleic acid. A complex between an effector protein and a guide nucleic acid can include multiple effector proteins or a single effector protein In some embodiments, the effector protein modifies the target nucleic acid when the complex contacts the target nucleic acid In someembodiments, the effector protein does not modify the target nucleic acid, but it is fused to a fusion partner protein that modifies the target nucleic acid when the complex contacts the target nucleic acid. A non-limiting example of an effector protein modify ing a target nucleic acid is cleaving of a phosphodiester bond of the target nucleic acid. Additional examples of modifications an effector protein can make to target nucleic acids are described herein and throughout. Herein, reference to an effector protein includes reference to a nucleic acid encoding the effector protein, unless indicated otherwise.

[0067] The term, “genetic disease,” as used herein, refers to a disease, disorder, condition, or syndrome associated with or caused by one or more mutations in the DNA of an organism having the genetic disease.

[0068] The term, “guide nucleic acid,” as used herein, refers to at least one nucleic acid comprising: a first nucleotide sequence that complexes to an effector protein on either the 5’ or 3’ terminus and the first nucleotide sequence can be fused to a second nucleotide sequence that hybridizes to a target nucleic acid. The first sequence may be referred to herein as a repeat sequence or guide sequence. The second sequence may be referred to herein as a spacer sequence. A guide nucleic acid may be referred to interchangeably with the term, “guide RNA, ” It is understood that guide nucleic acids may comprise DNA, RNA, or a combination thereof (e.g., RNA with a thymine base). Guide nucleic acids may include a chemically modified nucleobase or phosphate backbone.

[0069] The term, “extended guide RNA (rtgRNA),” as used herein refers to a single nucleic acid molecule comprising (not necessarily in the following order) (1 ) a guide RNA comprising (a) a protein binding sequence and (b) a spacer sequence: (2) optionally, a linker; and (3) a template RNA (retRNA) comprising (a) a primer binding sequence and (b) a template sequence. In some embodiments, the orientation of the rtgRNA from 5’ to 3’ is: guide nucleic acid, optional linker, and template RNA. In some embodiments, the orientation of the rtgRNA from 5’ to 3’ is: template RNA. linker, and guide RNA. It is understood that extended guide RNAs may comprise DN A, RNA, or a combination thereof (e.g., RNA with a thymine base).

[0070] The term “template RNA (retRNA)” as used herein, refers to anucleic acid comprising: a primer binding sequence and a template sequence. It is understood that template RNAs may comprise DNA, RNA. or a combination thereof (e.g., RNA with a thymine base). In some embodiments, the template RNA is linked to a guide RNA via a linker sequence to form an rtgRNA.

[0071] The terms “template sequence” and “RT template” as used herein, ref ers to a portion of a retRNA that contains a desired nucleotide modification relative to a target sequence or portion thereof. By way of non-limiting example, the desired edit may comprise one or more nucleotide insertions, deletions or substitutions relative to a target sequence or portion thereof. In some embodiments, it is identical to, complementary to, or reverse complementary to a target sequence or portion thereof. In some embodiments, the template sequence is complementary' to a sequence of the target nucleic acid that is adjacent to a nick site of a target site to be edited, with the exception that it includes a desired edit The template sequence (also referred in some embodiments as the RT template) can be complementary' to at least a portion of the target sequence with the exception of at least one nucleotide.

[0072] The terms, “primer binding sequence (PBS),” as used herein, refer to a portion of a retRNA and serves to bind to a primer sequence of the target nucleic acid. In some embodiments, the primer binding sequence binds to a primer sequence m the target nucleic acid that is formed after the target nucleic acid is cleaved by an effector protein. In some embodiments, the primer binding sequence is linked to the 3’ end of an retRNA In some embodiments, the primer binding sequence is located at the 5" end of a retRNA.

[0073] “Primer sequence” as used herein refers to a portion of the target nucleic acid that is capable of hybridizing wiith the primer binding sequence portion of an retRNA that is generated after cleavage of the target nucleic acid by an effector protein described herein .

[0074] The term “handle sequence,” as used herein, refers to a sequence that binds non- covalently with an effector protein. A handle sequence may also be referred to herein as a “scaffold sequence”. In some embodiments, the handle sequence comprises ail. or a portion of, a repeat sequence. Tn general, a single guide nucleic acid, also referred to as a single guide RNA (sgRNA), comprises a handle sequence that is capable of being non-covalently bound by’ an effector protein. The nucleotide sequence of a handle sequence may contain a portion of a tracrRNA, but generally does not comprise a sequence that hybridizes to a repeat sequence, also referred to as a repeat hybridization sequence.|0075] The term “trans-activating RNA (tracrRNA),” as used herein, refers to a nucleic acid that comprises a first sequence that is capable of being non-covalently bound by an effector protein, and a second sequence that hybridizes to a portion of a crRNA, which may be referred to as a repeat hybridization sequence.

[0076] The terms, “CRISPR RNA” or “crRNA,” as used herein, refer to a type of guide nucleic acid, wherein the nucleic acid is RNA comprising a first sequence, often referred to herein asa spacer sequence, that hybridizes to a target sequence of a target nucleic acid, and a second sequence, often referred to herein as a repeat sequence or guide sequence, that interacts with an effector protein. In some embodiments, the second sequence is bound by the effector protein. In some embodiments, the second sequence hybridizes to a portion of a tracrRNA, wherein the tracrRNA forms a complex with the effector protein

[0077] The term, “extension,” as used herein refers to additional nucleotides added to a nucleic acid, RNA, or DNA, or additional amino acids added to a peptide, polypeptide, or protein. Extensions may be processed during the formation of the guide RNA. In some embodiments, the extension comprises or consists of a template RNA.

[0078] By "hybridizable" or "complementary " or "substantially complementary" it is meant that a nucleic acid (e.g. RNA, DNA) comprises a sequence of nucleotides that enables it to noncovalently bind, i.e, form Watson-Crick base pairs and / or G / U base pairs, "anneal", or "hybridize." to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. Standard Watson-Crick basepairing includes: adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C). In addition, for hybridization between two RNA molecules (e.g., dsRNA), and for hybridization of a DNA molecule with an RNA molecule (e.g., when a DNA target nucleic acid base pairs with a guide RNA, etc ): guanine (G) can also base pair with uracil (U). For example, G / U base-pairing is at least partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon basepairing with codons m mRNA. Thus, in the context of this disclosure, a guanine (G) (e.g.. of dsRNA duplex of a guide RNA molecule; of a guide RNA base pairing with a target nucleic acid, etc.) is considered complementary' to both a uracil (U) and to an adenine (A). For example, when a G / U base-pair can be made at a given nucleotide position of a dsRNA duplex of a guide RNA molecule, the position is not considered to be non-complementary, but is instead considered to be complementary'.

[0079] The conditions of temperature and ionic strength determine the "stringency" of the hybridization. Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible. The conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art The greater the degree of complementarity between two nucleotide sequences, the greater the value of the meltingtemperature (Tm) for hybrids of nucleic acids having those sequences For hybridizations between nucleic acids with short stretches of complementarity (eg., complementarity over 35 or less, 30 or less, 25 or less, 22 or less, 20 or less, or 18 or less nucleotides) the position of mismatches can become important (see Sambrook et al.. Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001) at 1 1 .7-1 1 .8) Typically, the length for a hybridizable nucleic acid is 8 nucleotides or more (eg., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more. 25 nucleotides or more, or 30 nucleotides or more). Temperature, wash solution, salt concentration, and other conditions may be adjusted as necessary according to factors such as length of the region of complementation and the degree of complementation.

[0080] While hybridization typically occurs between two nucleotide sequences that are complementary, mismatches between bases are possible. It is understood that two nucleotide sequences need not be 100% complementary to be specifically’ hybridizable, or for hybridization to occur. Moreover, a nucleotide sequence may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a bulge, a loop structure or hairpin structure, etc,). A polynucleotide can comprise 60% or more, 65% or more. 70% or more, 75% or more, 80% or more. 85% or more, 90% or more, 95% or more, 98% or more, 99% or more. 99.5% or more, or 100% sequence complementarity to a target region within the target nucleic acid sequence to which it will hybridize. For example, an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary7nucleotides. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method. Example methods include BLAST programs (basic local alignment search tools); BioPython (Cock et al (2009) Biopython: freely7available Python tools for computational molecular biology and bioinformatics. Bioinformatics. 25, 1422-1423); and PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park. Madison Wis.), e.g.. using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math.,1981 , 2, 482-489), and the like. Unless otherwise noted, the % complementarity of two sequences referenced herein is determined by the BLAST or BioPython programs.

[0081] The term, “heterologous,’' as used herein, with reference to at least two different polypeptide or polynucleotide sequences, means that the two different polypeptide or polynucleotide sequences are not found similarly connected to one another in a native nucleic acid or protein. A protein that is heterologous to the effector protein is a protein that is not co valently linked via an amide bond to the effector protein in nature. In some embodiments, a heterologous protein is not encoded by a species that encodes the effector protein. A guide nucleic acid may comprise a first sequence and a second sequence, wherein the first sequence and the second sequence are not found covalently linked via a phosphodiester bond in nature.Thus, the first sequence is considered to be heterologous with the second sequence, and the guide nucleic acid may be referred to as a heterologous guide nucleic acid.

[0082] The term “linked’" as used herein in reference to an amino acid or nucleic acid sequence refers to any covalent mechanism by which two amino acid sequences or nucleic acid sequences are connected to each other in sequence. For example, in some embodiments, two sequences are linked directly together by a covalent bond (e g., an amide bond or phosphodi ester bond). In some embodiments, two sequences are linked together by a peptide or nucleic acid linker.

[0083] 'The term, “linked amino acids” as used herein, refers to at least two amino acids linked by an amide bond or a peptide bond.|00S4] The term, “linker,"’ as used herein, refers to an amino acid sequence or nucleic acid sequence that links a first polypeptide to a second polypeptide or a first nucleic acid to a second nucleic acid.

[0085] The term, “modified target nucleic acid,” as used herein, refers to a target nucleic acid, wherein the target nucleic acid has undergone a modification, for example, after contact with an I., SR and / or effector protein. In some embodiments, the modification is an alteration in the sequence of the target nucleic acid. In some embodiments, the modified target nucleic acid comprises an insertion, deletion, or replacement of one or more nucleotides compared to the unmodified target, nucleic acid.

[0086] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein, refer to a polymeric form of amino acids. A polypeptide may include coded and non-coded ammo acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. Accordingly, polypeptides as describedherein may comprise one or more mutations, one or more sequence modifications, or both. A peptide generally has a length of 100 or fewer linked amino acids.

[0087] A polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences.Sequence identity’ can be determined in a number of different ways. Percent identity’ can be determined using any convenient method. Example methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Alischul et al., J. Mol. Biol., 1990, 215, 403-410: Zhang and Madden, Genome Res.. 1997, 7, 649-656). the Gap program (Wisconsin Sequence Analysis Package. Version 8 for Unix, Genetics Computer Group,University- Research Park, Madison Wis.), e g., using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981. 2, 482-489), and the like. Unless otherwise noted. % sequence identities as referenced herein are determined using the BLAST or BioPython programs.

[0088] A DNA sequence that "encodes" a particular RNA is a DNA nucleotide sequence that is transcribed into RNA. A DNA polynucleotide may encode an RNA (mRNA) that is translated into protein (and therefore the DNA and the mRNA both encode the protein), or a DNA polynucleotide may' encode an RNA that is not translated into protein (e.g. tRNA, rRNA. microRNA (miRNA), a "non-coding" RNA (ncRNA), a guide RNA, etc.).

[0089] A "protein coding sequence" or a sequence that encodes a particular protein or polypeptide, is a nucleotide sequence that is transcribed into mRNA (in the case of DNA) and is translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory? sequences

[0090] The terms "DNA regulatory' sequences." "control elements,” and "regulatory- elements," used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and / or regulate transcription of a non-coding sequence (e.g., guide RNA) or a coding sequence (e.g., RNA-guided endonuclease and the like) and / or regulate translation of an encoded polypeptide.

[0091] The terra, “promoter"’ or “promoter sequence,” is a DNA regulatory7region capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding or non-coding sequence. A transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase, can also be found in a promoter region.Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes. Various promoters, including inducible promoters, may be used to drive expression by the various vectors of the present disclosure.

[0092] Tire term, “protospacer adjacent motif (PAM),” as used herein, refers to a nucleotide sequence found in a target nucleic acid that directs an effector protein to modify the target nucleic acid at a specific location. In some embodiments, a PAM is required for a complex of an effector protein and a guide nucleic acid to hybridize to and modify the target nucleic acid. In some embodiments, the complex does not require a PAM to modify the target nucleic acid. One example of a PAM sequences is 5’-NTTN-3’, where N can be any nucleic acid.

[8093] The term “RNA-dependent DNA polymerase (RDDP).” as used herein, refers to a DNA polymerase that uses a single-stranded RNA as a template for the synthesis of a complementary DNA strand,

[0094] The term, “RuvC” domain as used herein refers to a region of an effector protein that is capable of cleaving a target nucleic acid, and in certain embodiments, of processing a pre- crRNA. In some embodiments, the RuvC domain is located near the C -terminus of the effector protein A single RuvC domain may comprise RuvC subdomains, for example a RuvCI subdomain, aRuvCII subdomain and a RuvCIII subdomain. The term “RuvC” domain can also refer to a “RuvC-like” domain. Various RuvC-like domains are known in the art and are easily identified using online tools such as InterPro (www.ebi.ac.uk / interpro / ). For example, a RuvC- like domain may be a domain which shares homology with a region of TnpB proteins of theIS605 and other related families of transposons.

[0095] The term, “nickase” as used herein refers to an enzyme that possess catalytic activity for single stranded nucleic acid cleavage of a double stranded nucleic acid. A nickase cleaves a phosphodiester bond between two nucleotides of only one strand of dsDNA.

[0096] The terms, “’nuclease” and “endonuclease” are used interchangeably herein to mean an enzyme which possesses catalytic activity for nucleic acid cleavage.

[0097] The term, “nuclease activity,” is used to refer to catalytic activ ity that results in nucleic acid cleavage (e.g., ribonuclease activity (ribonucleic acid cleavage), or deoxyribonuclease activity (deoxyribonucleic acid cleavage), etc.).

[8098] The term "naturally-occurring," "unmodified," or "wild type" as used herein as applied to a nucleic acid, a polypeptide, a cell, or an organism, refers to a nucleic acid, polypeptide, cell, or organism that is found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism that can be isolated from a source in nature is naturally occurring.

[0099] The terms, “non-naturally occurring’’ and “engineered,” as used herein, are used interchangeably and indicate the involvement of the hand of man or machine. The terms, when referring to a nucleic acid, nucleotide, protein, polypeptide, peptide or ammo acid, refers to a modification of that molecule (e.g., chemical modification, nucleotide sequence, or amino acid sequence) that is not present in the naturally molecule The terms, when referring to a composition or system described herein, refer to a composition or system having at least one component that is not naturally associated with the other components of the composition or system. By way of a non -limiting example, a composition may include an effector protein and a guide nucleic acid that do not naturally occur together. Conversely, and as a non-limiting further clarifying example, an effector protein or guide nucleic acid that is “natural,” “naturally- occurring,” or “found in nature” includes an effector protein and a guide nucleic acid from a cell or organism that have not been genetically modified by the hand of man or machine.

[0100] The term, “variant." is intended to mean a form or version of a protein that differs from the wild-type protein A variant may have a different function or activity relative to the wild- type protein.

[0101] The term, “sequence modification,” as used herein refers to a modification of one or more nucleic acid residues of a nucleotide sequence or one or more amino acid residue of an ammo acid sequence, such as chemical modification of one or more nucleobases; or chemical modifications to the phosphate backbone, a nucleotide, a nucleobase, or a nucleoside. Such modifications can be made to an effector protein amino acid sequence or guide nucleic acid nucleotide sequence, or any sequence disclosed herein (e.g., a nucleic acid encoding an effector protein or a nucleic acid that, when transcribed, produces a guide nucleic acid). Methods of modifying a nucleic acid or amino acid sequence are known One of ordinary skill in the art will appreciate that the sequence modification(s) may be located at any position(s) of a nucleic acid such that the function of the nucleic acid, protein, composition or system is not substantially decreased. Nucleic acids provided herein can be prepared according to any available technique including, but not limited to chemical synthesis, enzymatic synthesis, which is generally termed in vitro-transcription, cloning, enzymatic, or chemical cleavage, etc. In some embodiments, the nucleic acids provided herein are not uniformly modified along the entire length of the molecule. Different nucleotide modifications and / or backbone structures can exist at various positions within the nucleic acid.

[0102] The term, “nuclear localization signal (NLS),” as used herein, refers to an entity (e.g., peptide) that facilitates localization of a nucleic acid, protein, or small molecule to the nucleus, when present in a cell that contains a nuclear compartment.

[0103] A person of ordinary skill in the art w ould appreciate that referring to a “nucleotide(s)’’, and / or “nucleoside(s)”. in the context of a nucleic acid molecule having multiple residues, is interchangeable and describe the sugar and base of the residue contained in the nucleic acid molecule. Similarly, a skilled artisan could understand that linked nucleotides and / or linked nucleosides, as used in the context of a nucleic acid having multiple linked residues, are interchangeable and describe linked sugars and bases of residues contained in a nucleic acid molecule. When referring to a “nucleobase(s)’; or linked nucleobase, as used in the context of a nucleic acid molecule, it can be understood as describing the base of the residue contained in the nucleic acid molecule, for example, the base of a nucleotide, nucleosides, or linked nucleotides or linked nucleosides. A person of ordinary skill in the art when referring to nucleotides, nucleosides, and / or nucieobases would also understand the differences between RNA and DNA (generally the exchange of uridine for thymidine or vice versa) and the presence of nucleoside analogs, such as modified uridines, do not contribute to differences in identity' or complementarity among polynucleotides as long as the relevant nucleotides (such as thymidine, uridine, or modified uridine) have the same complement (e.g., adenosine for all of thymidine, uridine, or modified uridine; another example is cytosine and 5-methylcytosine, both of which have guanosine or modified guanosine as a complement). Thus, for example, the sequence 5’-AXG-3' where X is any modified uridine, such as pseudouridine, N1 -methyl pseudo uridine, or 5-methoxyuridine, is considered 100% identical to AUG in that both are perfectly complementary to the same sequence (5!-CAU-3’).

[0104] Thus, the term "recombinant" polypeptide does not necessarily refer to a polypeptide whose amino acid sequence does not. naturally occur. Instead, a. "recombinant" polypeptide is encoded by a recombinant non-naturally occurring DMA sequence, but the amino acid sequence of the polypeptide can be naturally occurring ("wild type") or non-naturally occurring (e.g., a variant, a mutant, etc. ). A recombinant polypeptide is the product of a process run by a human or machine.

[0105] A "vector" or "expression vector" is a nucleic acid replicon, such as plasmid, phage, virus, artificial chromosome, or cosmid, to which another nucleic acid segment, i.e., an "insert", may be atached so as to bring about the replication of the attached segment in a cell.

[0106] The term “viral vector,” as used herein, refers to a nucleic acid to be delivered into a host cell via a recombinantly produced virus or viral particle.

[0107] An "expression cassette" comprises a DNA coding sequence operably linked to a promoter. "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner For instance, a promoter is operably linked to a coding sequence (or the coding sequence can also be said to be operably linked to the promoter) if the promoter affects its transcription or expression.

[0108] The terms "recombinant expression vector," or '"DNA construct" are used interchangeably herein to refer to a DNA molecule comprising a vector and an insert. Recombinant expression vectors are usually generated for the purpose of expressing and / or propagating the insert(s), or for the construction of other recombinant nucleotide sequences. The inserts) may or may not be operably linked to a promoter sequence and may or may not be operably linked to DNA regulatory sequences.

[0109] A cell has been "genetically modified," "transformed," or "transfected" by exogenous DNA or exogenous RNA, e.g., a recombinant expression vector, when such DNA has been introduced inside the cell. The presence of the exogenous DNA results in permanent or transient genetic change. The transforming DNA may or may not be integrated (covalently linked) into the genome of the ceil. In prokaryotes, yeast, and mammalian ceils for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter ceils through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones that comprise a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations[1)110] Suitable methods of genetic modification (also referred to as "transformation") include e.g., viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEl)-mediated transfection, DEAE-dextran mediated transfection. liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery (see, e.g.. Panyam et al. Adv Drug Deliv Rev. 2012 Sep 13. pii: S0169- 409X(l 2)00283-9. doi: 10. 1016 / j.addr 2012 09.023), and the like.

[0111] The choice of method of genetic modification is generally dependent on the type of cell being transformed and the circumstances under which the transformation is taking place (e.g., in vitro, ex vivo, or in vivo). A general discussion of these methods can be found in Ausubel, et al.. Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995.

[0112] "Nuclease" and "endonuclease" are used interchangeably herein to mean an enzyme which possesses catalytic activity for nucleic acid cleavage (e.g., ribonuclease activity (ribonucleic acid cleavage), deoxyribonuclease activity (deoxyribonucleic acid cleavage), etc.).

[0113] By "cleavage domain," "active domain," or "nuclease domain" of a nuclease it is meant the polypeptide sequence or domain within the nuclease which possesses the catalytic activity for nucleic acid cleavage. A cleavage domain can be contained in a single polypeptide chain or cleavage activity can result from the association of two (or more) polypeptides. A single nuclease domain may consist of more than one isolated stretch of amino acids within a given polypeptide.|0114] The terms, ‘treatment” or ’treating,” as used herein, are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results m the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying, or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.

[0115] A '‘syndrome”, as used herein, refers to a group of symptoms which, taken together, characterize a condition.

[0116] The terms "individual," "subject," "host," and "patient," used interchangeably herein, refer to an individual organism, e.g., a mammal, including, but not limited to, murines, simians, humans, non-human primates, ungulates, felines, canines, bovines, ovines, mammalian farmanimals, mammalian sport animals, and mammalian pets. The subject may be a mammal. The subject may be a human The subject may be diagnosed or at risk for a disease

[0117] “Large Serine Recombinase (LSR)” refers to a site-specific recombinase that promotes recombination reactions between recombinase recognition sequences (RRS) iocated on a donor and target DNA nucleic acid sequences.

[0118] "Recombinase recognition sequence” or “RRS” refers to the nucleic acid sequences present in donor and target DNA nucleic acids recognized by an LSR described herein. Recombinase recognition sequences may also be referred to in the art as DNA attachment sites. LSR systems were originally described in the context of bacterial and phage integration systems. Therefore, the RRS in the donor nucleic acid is referred to as “attP” (e.g., phage attachment site, used interchangeably with “RRS-i”) and the RRS in the target nucleic acid is referred to as “atB” (e.g , bacterial attachment site, used interchangeably with “RRS-2”). The product of attP * attB recombination is the integration of the donor nucleic acid sequence into the target nucleic acid sequences, in which the donor nucleic acid sequence is flanked by two new recombination sites, attL (used interchangeably with “RRS-L”) and attR (used interchangeably with “RRS-R”), each containing half sites derived from attP and attB, See FIG. 1A-FIG. 2C.

[0119] The term “base editing enzyme.” as used herein, refers to a protein, polypeptide, or fragment thereof that is capable of catalyzing the chemical modification of a nucleobase of a deoxyribonucleotide or a ribonucleotide. Such a base editing enzyme, for example, is capable of catalyzing a reaction that modifies a nucleobase that is present in a nucleic acid molecule, such as DNA or RNA (single stranded or double stranded). Non-limiting examples of the type of modification that a base editing enzyme is capable of catalyzing includes converting an existing nucleobase to a different nucleobase, such as converting a cytosine to a guanine or thymine or converting an adenine to a guanine, hydrolytic deamination of an adenine or adenosine, or methylation of cytosine (e g., CpG, CpA, CpT or CpC). A base editing enzyme itself may or may not bind to the nucleic acid molecule containing the nucleobase.

[0120] The term “base editor,” as used herein, refers to a fusion protein comprising a base editing enzyme linked to an effector protein. The base editing enzyme may be referred to as a fusion partner. The base editing enzyme can differ from a naturally occurring base editing enzyme. It is understood that any reference to a base editing enzyme herein also refers to a base editing enzyme variant. The base editor is functional when the effector protein is coupled to a guide nucleic acid. The guide nucleic acid imparts sequence specific activity to the base editor.By way of non-limiting example, the effector protein may comprise a catalytically inactive effector protein. Also, by way of non-limiting example, the base editing enzyme may comprise deaminase activity

[0121] Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0122] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure

[0123] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein,[M24] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.Large Serine Recombinase Systems

[0125] In some embodiments, the present disclosure provides large serine recombinases, compositions comprising the same, and methods of use in gene editing.

[0126] In general, large serine recombinases (LSRs) catalyze precise rearrangement of DNA through site-specific recombination of small sequences of DNA. referred to herein as recombinase recognition sequences (RRSs) or attachment (att) sites. Recombination mediated by LSRs typical!)' requires only the recombinase protein and the presence of small RRSs in the donor and target nucleic acid sequences. LSR-mediated recombination is also highly directional, reversed in the presence of a single accessor)' protein called a recombination directionality factor (RDF), and requires no cofactors to bind or bend DNA. LSRs can catalyze recombination between RRS sites on linear or circular DNA substrates and, depending on the position and orientation of the RRS sites, integrate, excise or invert sections of DNA (See examples of DNA editing by LSRs in FIGs. 1A-C).|0I2.7| In the absence of accessory' factors. LSRs mediate unidirectional recombination between a first RRS site (RRS- 1) in the donor nucleic acid sequence and a second RRS site (RRS-2) in the target nucleic acid sequence, generating RRS-L (left) and RRS-R (right) sites flanking the 5’ and 3’ ends (respectively) of the integrated donor nucleic acid sequence.

[0128] A major advantage of LSRs over other emerging technologies is that there is no theoretical upper limit on the size of the donor DNA, with reports demonstrating successful 27-kb integration into mammalian cells with Bxbl (Duportet, X. et al. A platform for rapid prototyping of synthetic gene networks in mammalian cells. Nucleic .Acids Res. 42, 13440 - 13451 (2014)). Although these features make LSRs highly attractive genome editing tools, the practical application of existing recombinases has been limited by several factors, most notably their low integration efficiency.

[0129] Insertion of transgenes into target nucleic acid sequences using LSRs may offer a number of advantages, including but not limited to:(a) integration is mediated by one LSR without relying on host factors for DNA integration;(b) LSRs do not degrade DNA or need to be engineered to bind a specific DNA sequence (in contrast to methods that rely on homing endonucleases);(c) integration can be targeted to a specific locus known to have minimal positional effects on transgene expression, unlike the use of transposons and retroviruses; and(d) transgenes integrated by LSRs though RRS-1 * RRS-2 recombination cannot be inverted or remobilized without the presence of a cognate RDF.

[0130] In some embodiments, the present disclosure provides an LSR, or a nucleic acid encoding the same, wherein the LSR comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to an ammo acid sequence selected from SEQ ID NOs: 1-3918 and 9761 -9763. In some embodiments, the LSR comprises or consists of an amino acid sequence selected from SEQ ID NOs: 1-3918 and 9761-9763.Exemplary LSR Systems

[0131] In some embodiments, the present disclosure provides methods of modifying a target nucleic acid sequence using an LSR system described herein. Exemplary embodiments of combinations of system elements and methods of modification are described below.

[0132] In some embodiments, the system provided herein comprises the following elements: (a) an LSR or a nucleic acid encoding the same: and (b) a donor DNA vector comprising a first recombinase recognition sequence(RRS-1).

[0133] In such embodiments, the target nucleic acid comprises a second recombinase recognition sequence (RRS-2) that may be naturally occurring or may be introduced into the target nucleic acid sequence. The donor DNA vector may be a circular DNA. In such embodiments, the entirety of the donor DNA vector can be inserted into the target nucleic acid sequence. See e.g. , FIG. 2A.

[0134] In some embodiments, the system provided herein comprises the following elements:(a) an LSR or a nucleic acid encoding the same; and(b) a donor DNA vector comprising two recombinase recognition sequences (RRS- 1A and RRS-1B) flanking the 5’ and 3’ ends of a sequence of interest (SOI) (or gene of interest(GOI).

[0135] In some embodiments, the target nucleic acid comprises two recombinase recognition sequences (RRS-2A and RRS-2B) that flank the 5" and 3rend of the target nucleic acid sequence The donor DNA may be circular DNA. In such embodiments, the LSR mediates recombination between RRS-1 A x RRS-2A and RRS-1B x RRS-2B such that a portion of thetarget nucleic acid sequence is excised and replaced with the sequence of interest from the donor DNA vector See e.g., FIG. 2B.

[0136] In some embodiments, the system provided herein comprises:(a) a first LSR or a nucleic acid encoding the same;(b) a second LSR or a nucleic acid encoding the same; and(c) a donor DNA vector comprising two recombinase recognition sequences (RRS-1A and RRS-1B) flanking the 5" and 3 ' ends of a gene of interest (GOI)|0137] In some embodiments, the target nucleic acid comprises two recombinase recognition sequences (RRS-2A and RRS-2B) that flank the 5’ and 3' end of the target nucleic acid sequence. The donor DNA may be circular DNA. In such embodiments, the first LSR mediates recombination between RRS-1A x RRS-2A and the second LSR mediates recombination between RRS-1B x RRS-2B such that a portion of the target nucleic acid sequence is excised and replaced with the sequence of interest from the donor DNA vector See e.g. , FIG, 2C.

[0138] In some embodiments, the LSR comprises an ammo acid sequence that is at least 75%. at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NOs: 1-2523 and 9761-9763; the RRS- 1 comprises a nucleotide sequence that is at least 60%, at least 70%. at least 80%, at least 85%, at least 90%. at least 95%. or 100% identical to a sequence selected from SEQ ID NOs:6442- 8964. 9740-9760, and 9764-9791; and the RRS-2 comprises a nucleotide sequence that is at least 60%, at least 70%, at least 80%. at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 3919-6441, 9719-9739, and 9792-9819, wherein the recombinase. RRS-1 and RRS-2 are in the same row' of TABLE 1 or TABLE 2.

[0139] In some embodiments, the LSR comprises an ammo acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NOs: 3, 16, 18, 20, 350, 441, 454. 455, 569, 591. 620, 955, 1 105, 1112, 1 162, 1319. 1452, 1518, 1735, 1944, 1966, 2050, 2068, 2172, 9761 , 9762. and 9763; the R.RS-1 comprises a nucleotide sequence that is at least 60%, at least 70%, at least 80%. at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 9740, 9741, 9743-9760, 9765-9770, 9774-9781, and 9785-9791; and the RRS-2 comprises a nucleotide sequence that is at least 60%. al least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ IDNOs: 9719, 9720, 9722-9739, 9793-9798, 9802-9809, and 9813-9819, wherein the recombinase, RRS-1 and RRS-2 are in the same row of any one of TABLES 13-15.

[0140] In some embodiments, the target sequence is selected from a nucleotide sequence that is at least 80%, at least 90%. at least 95% or 100% identical to a sequence selected from SEQ ID NOs: 8965-9266. and wherein the target sequence is in the same row of TABLE 1 as the recombinase, RRS-1, and RRS-2.

[0141] In some embodiments, the LSR or nucleic acid encoding the same, and a donor DNA vector are combined in a single composition. In some embodiments, the nucleic acid encoding the recombinase further comprises the donor DNA or sequence of interest. Recombinase Recognition Sequences

[0142] In some embodiments, the present disclosure comprises system for the modification of a target nucleic acid comprising (a) a recombinase or a nucleic acid encoding a recombinase; and (b) a donor DNA comprising a first recombinase recognition sequence (RRS-1), and wherein the target nucleic acid comprises a second RRS (RRS-2)

[0143] In some embodiments, the RRS-1 and / or RRS-2 are less than about 250 nucleotides in length. In some embodiments, the RRS-1 and / or RRS-2 are less than about 250, 200, 150, 100, or 50 nucleotides in length. In some embodiments, the RRS-1 and / or RRS-2. are about 10, 20, 30, 40, 50, 60, 70, 80, 90. 100, 1 10. 120, 130, 140, 150, 160, 170, 180, 190, 200. 210, 220, 230, 240, or 250 nucleotides in length.

[0144] In some embodiments, the RRS-1 comprises a nucleic acid sequence that is at least60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 6442-8964, 9740-9760, and 9764-9791. In some embodiments, the RRS-1 comprises a nucleic acid sequence selected from SEQ ID NOs: 6442- 8964 In some embodiments, the RRS-1 consists of a nucleic acid sequence selected from SEQ ID NOs: 6442-8964, 9740-9760. and 9764-9791 .

[0145] In some embodiments, the RRS-2 comprises a nucleotide sequence that is at least 60%, at least 70%, at least 80%. at least 85%, at least 90%, at least 95%. or 100% identical to a sequence selected from SEQ ID NOs: 3919-6441, 9719-9739, and 9792-9819. In some embodiments, the RRS-2 comprises a nucleic acid sequence selected from SEQ ID NOs: 3919- 6441, 9719-9739, and 9792-9819. In some embodiments, the RRS-2 consists of a nucleic acid sequence selected from SEQ ID NOs: 3919-6441. 9719-9739, and 9792-9819.

[0146] In some embodiments, the RRS-1 comprises a nucleic acid sequence that is at least 60%, al least 70%, al least 80%, at least 85%, at least 90%, at least 95%, or 100% identical toa sequence selected from SEQ ID NOs: 6442-8964, 9740-9760, and 9764-9791 and the RRS- 2 comprises a nucleotide sequence that is at least 60%. at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 3919- 6441, 9719-9739, and 9792-9819, wherein the RRS-1 and RRS-2 are in the same row of TABLE 1 or TABLE 2. In some embodiments, the RRS-1 comprises a nucleic acid sequence selected from SEQ ID NOs: 6442-8964, 9740-9760. and 9764-9791 and the RRS-2 comprises a nucleotide sequence selected from SEQ ID NOs: 3919-6441, 9719-9739. and 9792-9819, wherein the RRS-1 and RRS-2 are in the same row of TABLE 1 or TABLE 2. In some embodiments, the RRS-1 consists of a nucleic acid sequence selected from SEQ ID NOs: 6442- 8964. 9740-9760, and 9764-9791 and the RRS-2 consists of a nucleotide sequence selected from SEQ ID NOs: 3919-6441 , 9719-9739, and 9792-9819, wherein the RRS-I and RRS-2 are in the same row of TABLE 1 or TABLE 2.|0147] In some embodiments, an RRS is naturally occurring in a target nucleic acid sequence.In some embodiments, an RRS is introduced into a target nucleic acid sequence. In some embodiments, one or more RRSs are introduced into the target nucleic acid sequence. In some embodiments, only one RRS is introduced into a target nucleic acid sequence. In some embodiments, two RRSs are introduced into a target nucleic acid sequence. In some embodiments, the one or more RRSs are introduced into the target nucleic acid sequence using the systems described herein. In some embodiments, the guide nucleic acids described herein comprise one or more RRSs.[0I48j In some embodiments, an RRS is introduced into a target nucleic acid sequence with a precision editing system described herein. In some embodiments, an RRS is introduced into a target nucleic acid sequence with a method of precision editing described herein. In some embodiments, the precision editing system comprises a retRNA. In some embodiments, the retRNA comprises an RRSs. In some embodiments, the template sequence of the retRNA comprises one or more RRSs.

[0149] In some embodiments, the RRS-1 and / or the RRS-2 sequence comprise a kmer. A “kmer” is a sequence that, for a given LSR, is in each of the RRS-1, RRS-2, RRS-R, and RRS- L sequences, and is the minimal sequence required for the recombinase uses to mediate DNA rearrangements. A kmer for a given I., SR can be identified by querying the genomic DNA near the prophage boundary1within 500 bp of the recombinase, identifying the largest duplicate sequence (the kmer). and then identifying the pseudo palindromic site surrounding the kmer site. This site can then be resolved into the predicted RRS-1 and RRS-2 motifs. An illustrativemethod to identify a prophage boundary is Phage _Fi nder (Fonts, D. Nucleic Acids Research 2006; 34:20 p 5839). An illustrative method to identify a kmer is disclosed in Wang R., et al. Sci Rep 2016; 6: 23934. Both references are herein incorporated by reference in their entireties.

[0150] In some embodiments, the LSR is LSR 004, (LSR protein ID 2955954; SEQ ID NO: 16) and the kmer is SEQ ID NO: 9820 In some embodiments, the LSR is LSR_005, (LSR protein ID 3530952; SEQ ID NO: 20) and the kmer is SEQ ID NO: 9821. In some embodiments, the LSR is LSR 007, (LSR protein ID 3564078; SEQ ID NO: 3) and the kmer is SEQ ID NO: 9822. In some embodiments, the LSR is LSR_012, (LSR protein ID 3577396; SEQ ID NO: 18) and the kmer is SEQ ID NO: 9823. In some embodiments, the LSR is LSR 020, (LSR protein ID 3132173; SEQ ID NO: 7) and the kmer is SEQ ID NO: 9824. In some embodiments, the LSR is LSR 037, (LSR protein ID 2955313, SEQ ID NO: 2050) and the kmer is SEQ ID NO: 9825. In some embodiments, the LSR is LSR_057, (LSR protein ID 3249940: SEQ ID NO: 1319) and the kmer is SEQ ID NO: 9826. In some embodiments, the LSR is LSR 078, (LSR protein ID 3281695: SEQ ID NO: 1944) and the kmer is SEQ ID NO: 9827 Tn some embodiments, the LSR is LSR_086, (LSR protein ID 3580517; SEQ ID NO:2172) and the kmer is SEQ ID NO: 9828. In some embodiments, the LSR is LSR_106, (LSR protein ID 3170531; SEQ ID NO: 569) and the kmer is SEQ ID NO: 9829. In some embodiments, the LSR is LSR 112, (LSR protein ID 2955577; SEQ ID NO: 1452) and the kmer is SEQ ID NO: 9830. In some embodiments, the LSR is LSR_121, (LSR protein ID 3551216; SEQ ID NO: 455) and the kmer is SEQ ID NO: 9831. In some embodiments, theLSR is LSR 123, (LSR protein ID 3550343; SEQ ID NO: 454) and the kmer is SEQ ID NO: 9832. In some embodiments, the LSR is LSR_132, (LSR protein ID 3471751 ; SEQ ID NO: 1518) and the kmer is SEQ ID NO: 9833. In some embodiments, the LSR is LSR_137, (LSR protein ID 3467144; SEQ ID NO: 1966) and the kmer is SEQ ID NO: 9834. In some embodiments, the LSR is LSR 142, (LSR protein ID 3567541 , SEQ ID NO: 11 12) and the kmer is SEQ ID NO: 9835. In some embodiments, the LSR is LSR_I50, (LSR protein ID 3537784; SEQ ID NO: 591) and the kmer is SEQ ID NO: 9836. In some embodiments, the LSR is LSR 175, (LSR protein ID 3531245; SEQ ID NO: 350) and the kmer is SEQ ID NO: 9837 In some embodiments, the LSR is LSR_232 / LSR_509, (LSR protein ID 3507550; SEQ ID NO: 441) and the kmer is SEQ ID NO: 9838. In some embodiments, the LSR is LSR_253, (LSR protein ID 3568126; SEQ ID NO: 620) and the kmer is SEQ ID NO: 9839. In some embodiments, the LSR is LSR_263, (LSR protein ID 3474531 ; SEQ ID NO: 1 105) and the kmer is SEQ ID NO: 9840 In some embodiments, the LSR is LSR_300 / LSR_699, (LSRprotein ID 3459461 ; SEQ ID NO: 2068) and the kmer is SEQ ID NO: 9841. Tn some embodiments, the LSR is LSR .625, (LSR protein ID 3139148: SEQ ID NO: 1735) and the kmer is SEQ ID NO: 9842. In some embodiments, the LSR is LSR 662, (LSR protein ID 3445527; SEQ ID NO: 1162) and the kmer is SEQ ID NO: 9843.Precisian Editing Systems

[0151] In some embodiments, the present disclosure systems for precision editing and uses thereof. In some embodiments, components of the system are provided in a single composition. Such systems may be referred to as precision editing systems. Precision editing systems may be useful for generating an RRS in a target nucleic acid. For example, a target nucleic acid may not initially contain an RRS that can be utilized by an LSR system. However, a precision editing sy stem described herein could be used to generate an RRS in the target nucleic acid, thereby making it possible to insert a donor nucleic acid at that site with an LSR. Systems, compositions, and methods disclosed herein may comprise an LSR or nucleic acid encoding the LSR, and a precision editing system; and uses thereof.

[0152] In some embodiments, precision editing systems comprise an RNA-dependent DNA polymerase (RDDP), an effector protein (e.g,, a CRISPR associated (Cas) protein), a guide RNA, and a template RNA. In some embodiments, the RDDP and the effector protein are covalently linked. In some embodiments, the guide RNA and template RNA (retRNA) are fused or linked as an extended guide RNArtgRNA). In some embodiments, the present disclosure provides methods of modifying target nucleic acids utilizing precision editing systems. See FIGs. 3-6 for non-limiting depictions of exemplary precision editing systems.

[0153] In some embodiments, precision editing systems comprise: (a) an effector protein or a nucleic acid encoding the effector protein: (b) an RNA-directed DNA polymerase (RDDP) or a nucleic acid encoding the RDDP; (c) a guide RNA or nucleic acid encoding the guide RNA, wherein the guide RNA comprises (i) a first region comprising a protein binding sequence, and (ii) a second region comprising a spacer sequence that hybridizes to a target sequence of a first strand of a double stranded DNA (dsDNA) target nucleic acid, wherein the first region is located 5’ of the second region; and (d) a template RN A (retRN A) or nucleic acid encoding the retRNA, wherein the retRNA comprises (i) a primer binding sequence (PBS), and (ii) a template sequence that hybridizes to the target sequence of a second strand of the dsDNA target nucleic acid. In some embodiments, the guide RNA is linked to the retRNA. In some embodiments, the template sequence is located 5’ of the PBS, optionally wherein the 3’ end of the PBS is linked to the 5’ end of the template sequence. In some embodiments, the retRNA iscircularized. In some embodiments, the template sequence comprises an RRS sequence or a sequence that is reverse complementary to an RRS. The RRS may be an RRS that is recognized by an LSR described herein. In some embodiments, the one or more RRSs is linked to a primer binding sequence. The guide nucleic acid may interact with an effector protein and target the effector protein to the desired location in the cell genome. The effector protein may nick a strand of the cell genome and the RDDP may incorporate the one or more RRSs of the guide nucleic acid into the nicked site. This provides an RRS site at the desired location of the cell genome.

[0154] In some embodiments, the RDDP is not fused to the effector protein. In some embodiments, the RDDP is fused to an aptamer binding protein, and the guide RNA and / or retRNA comprises an aptamer that is capable of being bound by the aptamer binding protein. A non-limiting example of an aptamer is an MS2 aptamer and a non-limiting example of a corresponding aptamer binding protein is an MS2 coat protein. Additional examples of such localizing systems are described by Chen et al., FEBS J. (2013) 280:3734-3754.

[0155] In some embodiments, the fusion protein described herein comprises an effector protein comprising an ammo acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 9267-9401 and an RDDP comprising an ammo acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 9402-9482 In some embodiments, the fusion protein described herein comprises an effector protein comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 9267- 9401 and an RDDP comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 9402-9482.

[0156] In some embodiments, the fusion protein described herein comprises an effector protein comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%. or 99% identical to an amino acid sequence selected from SEQ ID NOs: 9267-9401 and an RDDP comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9413. In some embodiments, the fusion protein described herein comprises an effector protein comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 9267-9401 and an RDDP comprising or consisting of the amino acid sequence of SEQ ID NO: 9413. In some embodiments, the fusion protein comprises an ammo acid sequence that is at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 9702 or 9707. In some embodiments, the fusion protein comprises or consists of SEQ ID NO: 9702 or 9707.

[0157] In some embodiments, the fusion protein described herein comprises an effector protein comprising an amino acid sequence that is at least 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. or 99% identical to an amino acid sequence selected from SEQ ID NOs: 9267-9401 and an RDDP comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9415. In some embodiments, the fusion protein described herein comprises an effector protein comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 9267-9401 and an RDDP comprising or consisting of the amino acid sequence of SEQ ID NO: 9415. In some embodiments, the fusion protein comprises an amino acid sequence that is at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9703 or 9708. In some embodiments, the fusion protein comprises or consists of SEQ ID NO: 9703 or 9708.

[0158] In some embodiments, the fusion protein described herein comprises an effector protein comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 9267-9401 and an RDDP comprising an amino acid sequence that is at least 90%, 91 %, 92%, 93%, 94%, 95%. 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9421. In some embodiments, the fusion protein described herein comprises an effector protein comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 9267-9401 and an RDDP comprising or consisting of the amino acid sequence of SEQ ID NO: 9421. In some embodiments, the fusion protein comprises an ammo acid sequence that is at least 90%. 91%. 92%. 93%. 94%. 95%. 96%, 97%, 98%. or 99% identical to SEQ ID NO: 9704 or 9709. In some embodiments, the fusion protein comprises or consists of SEQ ID NO: 9704 or 9709.

[0159] In some embodiments, the fusion protein described herein comprises an effector protein comprising an amino acid sequence that is at least 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 92.67-9401 and an RDDP comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9423. In some embodiments, the fusion protein described herein comprises an effector protein comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 9267-9401 and an RDDP comprising or consisting of the amino acid sequence of SEQ ID NO: 9423. In some embodiments, the fusion protein comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 9705 or 9710. In some embodiments, the fusion protein comprises or consists of SEQ ID NO: 9705 or 9710.

[0160] In some embodiments, the fusion protein described herein comprises an effector protein comprising an ammo acid sequence that is at least 90%. 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%. or 99% identical to an amino acid sequence selected from SEQ ID NOs: 9267-9401 and an RDDP comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9431. In some embodiments, the fusion protein described herein comprises an effector protein comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 9267-9401 and an RDDP comprising or consisting of the amino acid sequence of SEQ ID NO: 9431. In some embodiments, the fusion protein comprises an amino acid sequence that is at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9706 or 9711. In some embodiments, the fusion protein comprises or consists of SEQ ID NO: 9706 or 9711.Tethers

[0161] In some embodiments the effector protein is complexed with ah or part of a biological tether or a protein localization sequence. In some embodiments the RDDP is bound to the biological tether or protein localization sequence. In some embodiments the guide RNA is bound to an aptamer that is recognized by the biological tether protein10162] In some embodiments, the biological tether or protein localization sequence is MS2, Csy4 or lambda N protein. In some embodiments, the effector proteins are complexed with a biological tether, and comprises all or part of (e.g., DNA binding domain from) the MS2. coat protein, endoribonuclease Csy4, or the lambda N protein. These proteins can be used to recruit RNA molecules containing a specific stem-loop structure to a locale specified by the Type V effector protein guide RNA targeting sequences. For example, a Type V effector protein variant fused to MS2 coat protein, endoribonuclease Csy4, or lambda N can be used to recruit a long non-coding RNA (IncRNA) such as XIST or HOTAIR; see. e g., Keryer-Bibens et al.. Biol. Cell 100: 125- 138 (2008), that is linked to the Csy4, MS2 or lambda N binding sequence. Alternatively, the Csy4, MS2 or lambda N protein binding sequence can be linked to another protein, e.g., as described in Keryer-Bibens et al., supra, and the protein can be targeted to the dCpfl variant binding site using the methods and compositions described herein. In some embodiments, the Csy4 is catalytically inactive.

[0163] In some embodiments, the RDDP comprises a reverse transcriptase. In some embodiments, the RDDP comprises an amino acid sequence that is at least 75%, at least 80%,al least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 9402-9482.

[0164] In some embodiments, the effector protein nicks a strand of the target nucleic acid and the RDDP synthesizes new DMA off the nicked end, wherein the new DNA is complementary to the template sequence (RT template), thereby producing the desired genomic edit in the target nucleic acid. In some embodiments, the effector protein nicks the target strand. In some embodiments, the effector protein nicks the non-target strand. In some embodiments, the effector protein is a Cas protein. In some embodiments, the effector protein is a Type V effector protein In some embodiments, the effector protein comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 9267-9401 and 9698-9701 . Additional effector proteins described herein and throughout are useful in precision editing systems.

[6165] In some embodiments, the effector protein nicks both the target strand and the nontarget strand sequentially. By way of non-limiting example, the effector protein may nick the target strand first and facilitate, with an rtgRNA or retRNA, the introduction of a desired genomic edit. After editing, the effector protein, m combination with a guide RNA that targets the non-target strand, can nick the non-target strand. Tire cellular DNA repair mechanisms can then repair the non-target strand using the edited target strand as template.

[0166] In some embodiments, systems comprise (a) an I, SR comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%. at least 99%, or 100% identical to SEQ ID NOs: 1-3918 and 9761-9763; (b) an effector protein comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%. at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 9267-9401; and (c) an RDDP comprising an ammo acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%>, at least 99%, or 100% identical to SEQ ID NOs: 9402-9482.

[0167] In some embodiments, the systems provided herein comprise (a) a fusion protein described herein (or a polynucleotide encoding the same); and (b) an rtgRNA comprising a guide RNA and a retRNA. In some embodiments, the systems provided herein comprise an RDDP comprising (a) an amino acid sequence that is at least 90% or at least 95% identical to any one of SEQ ID NOs: 9402-9482; (b) an effector protein; and (c) an rtgRNA comprising a guide RNA and a retRNA.

[0168] In some embodiments, the systems provided herein comprise (a) a fusion protein described herein (or a polynucleotide encoding the same); (b) an rtgRNA comprising a guide RNA and a template RNA; and (c) a guide RNA. In some embodiments, the systems provided herein comprise an RDDP comprising (a) an amino acid sequence that is at least 90% or at least 95% identical to any one of SEQ ID NOs: 9402-9482: (b) an effector protein; (c) an rtgRNA comprising a guide RNA and a retRNA; and (d) a guide RNA.[G169] In some embodiments, the enzyme that is capable of catalyzing the modification of the target nucleic acid forms a complex with an extended guide RNA (rtgRNA). In some embodiments, the extended guide RNA comprises (not necessarily in this order): a first region (also referred to as a protein binding region, protein binding sequence, or a repeat sequence) that interacts with an effector protein; a second region comprising a spacer sequence that is complementary to a target sequence of a first strand of a target dsDNA molecule; a third region comprising a template sequence that is complementary to at least a portion of the target sequence on the non-target strand of the target dsDNA molecule with the exception of at least one nucleotide: and a fourth region comprising a primer binding sequence that hybridizes to a primer sequence of the target dsDNA molecule that is formed when target nucleic acid is cleaved. The third region or template sequence may comprise a nucleotide having a different nucleobase than that of a nucleotide at the corresponding position in the target nucleic acid when the template sequence and the target sequence are aligned for maximum identity. In some embodiments, there is a linker between any one of the first, second, third and fourth regions.In some embodiments, the linker comprises a nucleotide. In some embodiments, the linker comprises multiple nucleotides.

[0170] In some embodiments, the third and fourth regions are 5’ of the first and second regions.In some embodiments, the order of the regions of the extended guide RNA from 5’ to 3‘ is: third region, fourth region, first region, and second region In some embodiments, there is a linker between any one of the first, second, third and fourth regions. In some embodiments, there is a linker between the first and fourth regions. In some embodiments, the effector protein is fused to an RDDP. In some embodiments, the RDDP comprises a reverse transcriptase. See, e.g., FIG. 3. The effector protein may nick a strand of the target nucleic acid and the RDDP synthesizes new DNA off the nicked end, wherein the new DNA is complementary to the template sequence (RT template), thereby producing the desired genomic edit in the target nucleic acid. In some embodiments, the effector protein nicks the target strand. In some embodiments, the effector protein nicks the non-target strand.

[0171] In some embodiments, the third and fourth regions are 3’ of the first and second regions.In some embodiments, the order of the regions of the extended guide RNA from 5’ to 3' is: first region, second region, third region, and fourth region. In some embodiments, there is a linker between the second and third regions. In some embodiments, the effector protein is fused to an RDDP. In some embodiments, the RDDP comprises a reverse transcriptase. See, e.g. ,FIG. 4. The effector protein may nick a strand of the target nucleic acid and the RDDP synthesizes new DNA off the nicked end, wherein the new DNA is complementary to the template sequence (RT template), thereby producing the desired genomic edit in the target nucleic acid. In some embodiments, the effector protein nicks the target strand. In some embodiments, the effector protein nicks the non-target strand.

[0172] In some embodiments, compositions and systems comprise (1) a guide RNA comprising (a) a first region (also referred to as a protein binding region, protein binding sequence, or repeat sequence) that interacts with an effector protein and (b) a second region comprising a spacer sequence that is complementary to a target sequence of a first strand of a target dsDNA molecule; and (2) a template RNA (retRNA) comprising (a) a. primer binding sequence that hybridizes to a primer sequence of the target dsDNA molecule that is formed when the target nucleic acid is cleaved and (b) a template sequence that is complementary to at least a portion of the target sequence on the second strand of the target dsDNA molecule with the exception of at least one nucleotide. The template sequence may comprise a nucleoli de having a different nucleobase than that of a nucleotide at the corresponding position in the target nucleic acid when the template sequence and the target sequence are aligned for maximum identity In some embodiments, the primer binding sequence is linked to the template sequence. In some embodiments, the guide RNA and the template RNA are covalently connected see, e.g. FIGs. 3-4. In some embodiments, the guide RNA and the template RNA are not covalently connected, see, e.g. FIGs, 5A-5B and FIG. 6. In some embodiments, such compositions comprise an effector protein that is fused to a RDDP In some embodiments, such compositions comprise an effector protein that is not linked or fused (e.g . covalently linked) to an RDDP. Compositions and systems that comprise an RDDP that is not Imked / fused to an effector protein and / or a guide RNA that is not fused / linked to a template RNA may be referred to as a "‘split protein / RNA design" See, e.g. FIGs. 5A-5B and FIG. 6, The effector protein may nick a strand of the target nucleic acid and the RDDP synthesizes new DNA off the nicked end, wherein the new DNA is complementary to the template sequence (RT template), thereby producing the desired genomic edit in the target nucleic acid. In some embodiments, theeffector protein nicks the target strand In some embodiments, the effector protein nicks the non-target strand.Base editors

[0173] In some embodiments, the present disclosure systems for base editors and uses thereof. Base editors may be useful for generating an RRS in a target nucleic acid. For example, a target nucleic acid may not initially contain an RRS that can be utilized by an LSR system However, a base editor described herein could be used to generate an RRS in the target nucleic acid, thereby making it possible to insert a donor nucleic acid at that site with an LSR. Systems, compositions, and methods disclosed herein may comprise an LSR or nucleic acid encoding the LSR, and a base editor; and uses thereof.

[0174] In some embodiments, fusion partners edit anucleobase of a target nucleic acid. Fusion proteins comprising such a fusion partner and an effector protein may be referred to as base editors. Such a fusion partner may be referred to as a base editing enzyme. In some embodiments, a base editor comprises a base editing enzyme variant that differs from a naturally occurring base editing enzyme, but it is understood that any reference to a base editing enzyme herein also refers to a base editing enzyme van ant. In some embodiments, abase editor may be a fusion protein comprising a base editing enzyme linked to an effector protein. In some embodiments, the amino terminus of the fusion partner protein is linked to the carboxy terminus of the effector protein by the linker. In some embodiments, the carboxy terminus of the fusion partner protein is linked to the amino terminus of the effector protein by the linker. The base editor may be functional when the effector protein is coupled to a guide nucleic acid. The guide nucleic acid imparts sequence specific activity to the base editor. By wav of non-limiting example, the effector protein may comprise a catalytically inactive effector protein (e g., a catalytically inactive variant of an effector protein described herein), Also, by way of nonlimiting example, the base editing enzyme may comprise deaminase activity. Additional base editing enzymes are described herein.

[0175] In some embodiments, base editing enzymes are capable of catalyzing editing (e g., a chemical modification) of a nucleobase of a nucleic acid molecule, such as DMA or RNA (single stranded or double stranded). In some embodiments, a base editing enzyme, and therefore a base editor comprising the same, is capable of converting an existing nucleobase to a different nucleobase. such as: an adenine (A) to guanine (G); cytosine (C) to thymine (T); cytosine (C) to guanine (G); uracil (U) to cytosine (C); guanine (G) to adenine (A); hydrolytic deamination of an adenine or adenosine, or methylation of cytosine (e g., CpG, CpA, CpT orCpC). In some embodiments, base editors edit a nucleobase on a ssDNA. In some embodiments, base editors edit a nucleobase on both strands of dsDNA. In some embodiments, base editors edit a nucleobase of an RNA.

[0176] In some embodiments, a base editing enzyme itself may or may not bind to the nucleic acid molecule containing the nucleobase In some embodiments, upon binding to its target locus in the target nucleic acid (e.g., a DNA molecule), base pairing between the guide nucleic acid and target strand leads to displacement of a small segment of ssDNA in an “R-loop’r. In some embodiments, DNA bases within the R-loop are edited by the base editor having the deaminase enzyme activity. In some embodiments, base editors for improved efficiency in eukary otic cells comprise a catalytically inactive effector protein that may' generate a nick in the non-edited strand, inducing repair of the non-edited strand using the edited strand as a template.|f>177] In some embodiments, a base editor comprises a deaminase enzyme. Exemplary' deaminases are described in US20210198330. WO2021041945, WO2021050571 Al, and W02020123887, all of which are incorporated herein by reference in their entirety'. Exemplary deaminase domains are described WO 2018027078 and W02017070632, and each are hereby incorporated in its entirety by reference. Also, additional exemplary deaminase domains are described in Komor et al.. Nature, 533, 420-424 (2016): Gaudelli et al.. Nature, 551. 464-471 (2017); Komor et al., Science Advances, 3:eaao4774 (2017), and Rees et al., Nat Rev Genet, 2018 Dec;19(12):770-788. doi: 10.1038 / s41576-018-0059-l, which are hereby' incorporated by reference in their entirety. In some embodiments, the deaminase is ADAR1 / 2, ADAR-2, AID, or any functional variant thereof In some embodiments, the deaminase functions as a monomer. In some embodiments, the deaminase functions as heterodimer with an additional protein. In some embodiments, base editors comprise a DNA glycosylase inhibitor (e.g., an uracil glycosylase inhibitor (UGI) or uracil N-glycosylase (UNG)).

[0178] In some embodiments, a base editor is a cytosine base editor (CBE) and comprises a cytosine base editing enzyme. In some embodiments, the cytosine base editing enzyme may convert a cytosine to a thy mine. In some embodiments, a cytosine base editing enzyme may accept ssDNA as a substrate but may not be capable of cleaving dsDNA, as linked to a catalytically inactive effector protein. In some embodiments, when bound to its cognate DNA, the catalytically inactive effector protein of the CBE may perform local denaturation of the DNA duplex to generate an R-loop in which the DNA strand not paired with a guide nucleic acid exists as a disordered single-stranded bubble In some embodiments, the catalyticallyinactive effector protein generated ssDNA R-loop may enable the CBE to perform efficient and localized cytosine deamination in vitro. In some embodiments, deamination activity is exhibited in a window of about 4 to about 10 base pairs. In some embodiments, fusion to the catalytically inactive effector protein presents a target site to the cytosine base editing enzyme in high effective molarity, which may enable the CBE to deaminate cytosines located in a variety of different sequence motifs, with differing efficacies. In some embodiments, the CBE is capable of mediating RNA-programmed deamination of target cytosines in vitro or in vivo. In some embodiments, the cytosine base editing enzyme is a cytidine deaminase. In some embodiments, the cytosine base editing enzyme is a cytosine base editing enzyme described by Koblan et al. (2018) Nature Biotechnology' 36:848-846; Komor et al (2016) Nature 533:42.0-424; Koblan et al. (2021) “Efficient OG-to-G*C base editors developed using CRISPRi screens, target-library analysis, and machine learning,’" Nature Biotechnology; Kurt et al. (2021) Nature Biotechnology 39:41-46; Zhao et al. (2021) Nature Biotechnology 39:35-40; and Chen et al. (2021 ) Nature Communications 12: 1384, all incorporated herein by reference.

[0179] In some embodiments, CBEs comprise a uracil glycosylase inhibitor (UGI) or uracil N- glycosylase (UNG). In some embodiments, base excision repair (BER) of U»G in DNA is initiated by a UNG, which recognizes a U»G mismatch and cleaves the gly osidic bond between a uracil and a deoxyribose backbone of DNA. In some embodiments, BER results in the reversion of the U»G intermediate created by the first CBE back to a C»G base pair. In some embodiments, the UNG may be inhibited by fusion of a UGI. In some embodiments, the CBE comprises a UGI. In some embodiments, a C -terminus of the CBE comprises the UGI. In some embodiments, the UGI is a small protein from bacteriophage PBS. In some embodiments, the UGI is a DNA mimic that potently inhibits both human and bacterial UNG In some embodiments, the UGI inhibitor is any protein or polypeptide that inhibits UNG. In some embodiments, the CBE may mediate efficient base editing in bacterial cells and moderately efficient editing in mammalian cells, enabling conversion of a C«G base pair to a T» A base pair through a UsG intermediate. In some embodiments, the CBE is modified to increase base editing efficiency while editing more than one strand of DNA.

[0180] In some embodiments, a CBE nicks a non-edited DNA strand. In some embodiments, the non-edited DNA strand nicked by the CBE biases cellular repair of a U«G mismatch to favor a U*A outcome, elevating base editing efficiency. In some embodiments, a APOBEC1- nickase-UGI fusion efficiently edits in mammalian cells, while minimizing frequency of nontarget indels. In some embodiments, base editors do not comprise a functional fragment of thebase editing enzyme. Tn some embodiments, base editors do not comprise a functional fragment of a UGI, where such a fragment may be capable of excising a uracil residue from DNA by cleaving an N-glycosidic bond.

[0181] In some embodiments, the base editor further comprises a non-protein uracil-DNA glycosylase inhibitor (npUGI). In some embodiments, the npUGI is selected from a group of small molecule inhibitors of uracil-DNA glycosylase (UDG), or a nucleic acid inhibitor of UDG. In some embodiments, the npUGI is a small molecule deri ved from uracil. Examples of small molecule non-protein uracil-DNA glycosylase inhibitors, fusion proteins, and Cas- CR1SPR systems comprising base editing activity are described in WO2021087246. which is incorporated by reference in its entirety.

[0182] In some embodiments, the cytosine base editing enzyme, is a cytidine deaminase. In some embodiments, the cytidine deaminase base editing enzy me is generated by ancestral sequence reconstruction as described in WO2019226953, which is hereby incorporated by reference in its entirety. Non-limiting exemplary' cytidine deaminases suitable for use with effector proteins described herein include: APOBEC1, APOBEC2, APOBEC3C, APOBEC3D,APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, APOBEC3A, BE! (APOBEC 1 -XTEN- dCas9), BE2 (APOBEC l -XTEN-dCas9-UGI). BE3 (APOBEC !-XTEN-dCas9(A840H)-UGI), BE3-Gam. saBE3, saBE4-Gam, BE4, BE4-Gam, saBE4. and saBE4-Gam as described in WO2021 163587. WO2021087246, WO2021062227, and WO2020123887, which are incorporated herein by reference in their entirety.

[0183] In some embodiments, a base editor is a cytosine to guanine base editor (CGBE) and comprises a cytosine to guanine base editing enzyme. A CGBE may convert a cytosine to a guanine.

[0184] In some embodiments, a base editor is an adenine base editor (ABE) and comprises an adenine base editing enzyme. An ABE may convert an adenine to a guanine. In some embodiments, an ABE converts an A*T base pair to a G*C base pair. In some embodiments, the ABE converts a target A»T base pair to G»C in vivo or in vitro. In some embodiments. ABEs provided herein reverse spontaneous cytosine deamination, which has been linked to pathogenic point mutations. In some embodiments, ABEs provided herein enable correction of pathogenic SNPs (-47% of disease-associated point mutations). In some embodiments, the adenine comprises exocyclic amine that has been deaminated (e.g., resulting in altering its base pairing preferences). In some embodiments, deamination of adenosine yields inosine In some embodiments, inosine exhibits the base-pairing preference of guanine in the context of apolymerase active site, although inosine in the third position of a tRNA anticodon is capable of pairing with A, U, or C in mRNA during translation. Non-limiting exemplary adenine base editing enzymes suitable for use with effector proteins described herein include: ABE8e, ABE8.20m, AP0BEC3A. Anc APOBEC (a.k.a. AncBE4Max), and BtAPOBEC2. Non- limiting exemplary' ABEs suitable for use herein include: ABE7. ABE8.1m, ABES.2m.ABE8.3m, ABE8.4m, ABE8.5m, ABE8 6m, ABE8.7m, ABES.8m, ABE8.9m, ABE8.10m, ABE8.11m, ABES. 12m, ABES.13m, ABES.14m, ABE8.15m, ABE8.16m, ABE8.17m, ABE8. 18m, ABES. 19m, ABES.20m, ABES.2 Im, ABE8.22m, ABE8.23m, ABE8.24m, ABES. Id, ABE8.2d. ABES, 3d, ABE8 4d. ABE8.5d. ABE8.6d, ABE8.7d. ABE8.8d, ABE8.9d. ABE8.10d, ABE8.1 Id. ABE8.12d. ABE8.13d, ABE8 14d, ABE8.15d, ABE8.16d, ABE8.17d,ABE8.18d, ABE8.19d, ABE8.20d, ABE8.2M, ABE8.22d, ABES.23d, and ABE8.24d. In some embodiments, the adenine base editing enzyme is an adenine base editing enzyme described in Chu et al., (2021) The C RISER Journal 4:2: 169-177, incorporated herein by reference. In some embodiments, the adenine deaminase is an adenine deaminase described by Koblan et al. (2018) Nature Biotechnology 36:848-846, incorporated herein by reference. In some embodiments, the adenine base editing enzyme is an adenine base editing enzyme described by Tran et al. (2020) Nature Commrmications 1 1 :4871.

[0185] In some embodiments, the base editing enzyme alters a base within a nucleotide sequence that is not a recombinase recognition sequence into a nucleotide sequence that is a recombinase recognition sequence. The nucleotide sequence that is a recombinase recognition sequence is a sequence selected from SEQ ID NOs: 3919-8964, 9719-9760. and 9764-9819. In some embodiments, the base editing enzyme alters one base of the nucleotide sequence io make the nucleotide sequence into a recombinase recognition sequence In some embodiments, the base editing enzy me alters two or more bases of the nucleotide sequence to make the nucleotide sequence into a recombinase recognition sequence. In some embodiments, multiple base editing enzymes are used to alter a nucleotide sequence that is not a recombinase recognition sequence into a nucleotide sequence that is a recombinase recognition sequence.Effector Proteins|0186] Provided herein, are compositions, systems, raid methods comprising an effector protein and uses thereof In general, the effector protein comprises a CRISPR associated (Cas) protein. In some embodiments, the Cas protein comprises a Type II Cas protein. In some embodiments, the Cas protein does not comprise a Type II Cas protein. In some embodiments, the effector protein does not comprise a Cas9 protein In some embodiments, the Cas proteincomprises a Type V Cas protein In some embodiments, the Type V Cas protein comprises an RuvC domain and does not comprise an HNH domain. In some embodiments, the Cas protein comprises a Type VU-3 Cas protein. In some embodiments, the Cas protein comprises a Type VU-4 protein. In some embodiments, the Cas protein comprises a CasPhi (Cas®, Casl2J) protein In some embodiments, the Cas protein comprises a Cas 14 protein. In some embodiments, the Casl4 protein is a Casl4a protein. In some embodiments, the Casl4 protein is a Casl4b protein. In some embodiments, the Cas protein is CasM.265466.

[0187] In general, effector proteins described herein comprise one or more functional domains. Effector protein functional domains may include a protospacer adjacent motif (PAM)- interacting domain, an oiigonucleotide-mteracting domain, one or more recognition domains, a non-target strand interacting domain, and a RuvC domain. A PAM interacting domain can be a target strand PAM interacting domain (TPID) or anon-target strand PAM interacting domain (NTPID). In some embodiments, a PAM interacting domain, such as a TPID or a NTPID, on an effector protein descri bes a region of an effector protein that interacts with target nucleic acid. In some embodiments, the effector proteins comprise a RuvC domain. In some embodiments, a RuvC domain comprises with substrate binding activity, catalytic activity, or both. In some embodiments, the RuvC domain may be defined by a single, contiguous sequence, or a set of RuvC subdomains that are not contiguous with respect to the primary amino acid sequence of the protein. An effector protein of the present disclosure may include multiple RuvC subdomains, which may combine to generate a RuvC domain with substrate binding or catalytic activity. For example, an effector protein may include three RuvC subdomains (RuvC-I, RuvC-II, and RuvC-III) that are not contiguous with respect to the primary' amino acid sequence of the effector protein, but form a RuvC domain once the protein is produced and folds. In some embodiments, effector proteins comprise one or more recognition domain (REC domain) with a. binding affinity for a guide nucleic acid or for a guide nucleic acid-target nucleic acid heteroduplex. An effector protein may comprise a zinc finger domain. In some embodiments, the effector protein does not comprise an HNH domain.

[0188] In some embodiments, an effector protein is similar to a naturally occurring effector protein The effector protein may lack a portion of the naturally occurring effector protein. The effector protein may comprise a mutation relative to the naturally-occurring effector protein, wherein the mutation is not found in nature. The effector protein may also comprise at least one additional amino add relative to the naturally-occurring effector protein. For example, the effector protein may comprise an addition of a nuclear localization signal relative to the naturaloccurring effector protein. In some embodiments, a nucleotide sequence encoding the effector protein is codon optimized (<?.g., for expression in a eukaryotic cell) relative io the naturally occurring sequence.

[0189] An effector protein may be brought into proximity of a target nucleic acid in the presence of a guide nucleic acid when the guide nucleic acid includes a nucleotide sequence that is complementary with a target sequence in the target nucleic acid. The ability of an effector protein to modify a target nucleic acid may be dependent upon the effector protein being bound to a guide nucleic acid and the guide nucleic acid being hybridized to a target nucleic acid. An effector protein may also recognize a protospacer adjacent motif (PAM) sequence present in the target nucleic acid, which may direct the modification activity- of the effector protein. An effector protein may have nuclease activity, including nickase activity'.

[0190] In some embodiments, effector proteins are compact, which may' be beneficial for therapeutic delivery'. The compact nature of these effector proteins max allow for them to be more easily packaged and delivered with higher efficiency. In some embodiments, the length of the effector protein is not greater than 700 linked amino acid residues. In some embodiments, the length of the effector protein is not greater than 750 linked amino acid residues. In some embodiments, the length of the effector protein is not greater than 800 linked amino acid residues. In some embodiments, the length of the effector protein is not greater than 850 linked amino acid residues. In some embodiments, the length of the effector protein is not greater than 900 linked ammo acid residues. In some embodiments, the length of the effector protein is at least 300, at least 325. at least 350, at least 375, or at least 400 linked amino acid residues. In some embodiments, the length of the effector protein is about 350 to about 450 linked amino acid residues In some embodiments, the length of the effector protein is about 375 to about 475 linked amino acid residues. In some embodiments, the length of the effector protein is about 400 to about 450 linked amino acid residues. In some embodiments, the length of the effector protein is about 400 to about 500 linked amino acid residues. In some embodiments, the length of the effector protein is about 350 to about 400, about 400 to about 450, about 450 to about 550, about 400 to about 420, about 420 to about 440, about 440 to about 460, about 460 to about 480, about 480 to about 500, about 500 to about 520, about 520 to about 540, about 540 to about 560, about 560 to about 580, about 580 to about 600, about 600 to about620, about 620 to about 640, about 640 to about 660, about 660 to about 680, about 680 to about 700 linked amino acids. In some embodiments, the length of the effector protein is at least 200, at least 225, at least 250, at least 275 al least 300, at least 325, at least 350, at least375, at least 400, at least 425, at least 450, at least 475, at least 500, at least 525, at least 550, at least 575, at least 600, at least 625. at least 650, at least 675, at least 700, at least 725, at least 750, at least 775 linked amino acids. In some embodiments, the length of the effector protein is about 700 to about 800 linked amino acid residues. In some embodiments, the length of the effector protein is about 750 to about 850, about 700 to about 820, about 720 to about 840, about 740 to about 860, about 760 to about 880, about 780 to about 900, about 800 to about 920, about 820 to about 940, about 840 to about 960, about 860 to about 980, about 880 to about 1000, about 900 to about 1020, about 920 to about 1040, about 940 to about 1060, about 960 to about 1080, about 980 to about 1100 linked amino acids. In some embodiments, the length of the effector protein is about 1000 to about 1100 linked amino acid residues

[0191] TABLE 4 provides illustrative amino acid sequences of effector proteins, guide sequences, and PAM sequences that are useful in the compositions, systems and methods described herein. With regards to the PAM sequences: N is any nucleotide. V is A. C, or G; B is C, G, or T; and S is G or C. In certain embodiments, compositions, systems, and methods provided herein comprise an effector protein and an engineered guide nucleic acid, wherein the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%. at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to any one of the sequences as set forth in TABLE 4. In some embodiments, effector proteins differ from a sequence in TABLE 4 by one or more amino acids. In some embodiments, effector proteins differ from a sequence in TABLE 4 by 1 amino acid, 2 amino acids. 3 amino acids, 4, amino acids, 5 amino acids, 6 ammo acids, 7 ammo acids. 8 amino acids. 9 ammo acids or 10 ammo acids. In some embodiments up to 15 amino acids are modified In some embodiments up to 20 amino acids are modified. In some embodiments up to 25 amino acids are modified.

[0192] In some embodiments the modifications are conservative substitutions relative to the effector protein sequence in TABLE 4. In some embodiments, the amino acids that differ from the effector proteins are non-conservative substitutions relative to the effector protein sequence in TABLE 4. In some embodiments, a mutation may affect the catalytic activity of the effector protein and results in a catalytically reduced or catalytically inactive mutant. In some embodiments, a mutation can result in the effector protein having nickase activity or increased nickase activity. In some embodiments, a mutation can result in the effector protein having reduced or no nuclease activity' but gaming nickase activity.

[0193] TABLE 4 provides sequences for exemplary CasPhi (CasO) proteins which may process their associated crRNA array and cleave the array into individual crRNAs. Engineered gRNAs in which extra nucleotides are added to the sequence (i.e., RT template) on the 5' end, may be cleaved off by a CasPhi. This contrasts with Cas9, which does not process its own crRNA array and therefore is not subject to the same issue.

[0194] In some embodiments, the effector protein is CasM.265466 (SEQ ID NO: 9401) or a variant thereof described herein. In some embodiments, systems and compositions comprise an effector protein and a guide nucleic acid, wherein the effector protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%. at least 90%. at least 95%. at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9401, and the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%. or 100% identical to the sequence:ACAGCUUAUUIJGGAAGCUGAAAUGUGAGGUIJIJAUAACACUCACAAGAAIJCCIJ GAAAAAGGAUGCCAAAC (SEQ ID NO: 9696). In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%. at least 90%. at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9694.

[0195] In some embodiments, the effector protein is CasM. 19952 (SEQ ID NO: 9338) or a variant thereof described herein. In some embodiments, systems and compositions comprise an effector protein and a guide nucleic acid, wherein the effector protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%. at least 90%. at least 95%. at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9338, and the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, ai least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%. or 100% identical to the sequence:U GGGGC AGU U GGU U GC CC UU AGC C U GAGGC A U U U A U L!GC AC UCGGGA AGU AC CAUUUCUCAGAAAAAGGAUGCCAAAC (SEQ ID NO: 9697). In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or100% identical to a sequence selected from SEQ ID NO: 9548-9567.

[0196] In some embodiments, the effector protein is CasPhi. 12 (SEQ ID NO: 9278) or a variant thereof described herein In some embodiments, systems and compositions comprisean effector protein and a guide nucleic acid, wherein the effector protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9278, and the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%. at least 97%, at least 98%, at least 99%, or 100% identical to any one of sequence: GUAGAL'UGCUCCUUACGAGGAGAC (SEQ ID NO: 9714); CUUUCAAGACUAAUAGAUUGCUCCUUACGAGGAGAC (SEQ ID NO: 9488);AUAGAUUGCUCCUUACGAGGAGAC (SEQ ID NO: 9716);U AGA U U GC I CC U U ACGAGGAGAC (SEQ ID NO: 9717); AGAUUGCUCCUUACGAGGAGAC (SEQ ID NO: 9718);GAUUGCUCCUUACGAGGAGAC (; i»EQ ID NO: 9715); orAUUGC UCCUUACGAGGAGAC (SEQ ID NO: 9489).Protospacer Adjacent Motif (PAM)

[0197] Effector proteins of the present disclosure, dimers thereof, and niultimeric complexes thereof, may cleave or nick a target nucleic acid within or near a protospacer adjacent motif (PAM) sequence of the target nucleic acid. In some embodiments, cleavage occurs within 1, 2, 3. 4. 5, 6. 7, 8, 9. 10, I L 12. 13, 14, 15, 16, 17, 18, 19. 20, 21, 22, 23, 24, or 25 nucleotides of a 5’ or 3’ terminus of a PAM sequence In some embodiments, a target nucleic acid may comprise a PAM sequence adjacent to a target sequence that is complementary to a guide nucleic acid spacer region. PAMs in compositions, systems, and methods herein are further described throughout the application. In some embodiments, the PAM is located 5’ of the target sequence on the non-target strand of the target nucleic acid. In some embodiments, the PAM is located 3’ of the target sequence on the target strand of the target nucleic acid

[0198] In some embodiments, a target nucleic acid comprises a target sequence that is adjacent to a PAM sequence, wherein the PAM sequence comprises any one of the PAM sequences as set forth in TABLE 4. In some embodiments, systems, compositions, and / or methods described herein comprise a target nucleic acid comprising a target sequence that is adjacent to a PAM sequence, wherein the PAM sequence comprises any one of the PAM sequences as set forth in TABLE 4. RNA-dependent DNA polymerases (RDDP)

[0199] In some embodiments, the present disclosure provides for systems, compositions, and methods comprising an RNA-dependent DNA polymerase (RDDP) or a use thereof. Herein,RDDPs are enzymes that are capable of generating a DNA polynucleotide from an RNA template polynucleotide In some embodiments, the RDDP is a reverse transcriptase. A nonlimiting example of a reverse transcriptase is a Moloney murine leukemia virus (M-MLV) reverse transcriptase (e.g., SEQ ID NO: 9471 or 9472). Herein, the term RDDP encompasses functional domains thereof. In some embodiments, the functional domain is a polymerase domain, an RNAse domain, or a combination thereof.19200] In some embodiments, the RDDP comprises less than 800 amino acids. In some embodiments, the RDDP comprises less than 800, less than 700, less than 600, less than 500, less than 400. less than 300 ammo acids. In some embodiments, the RDDP comprises at least 200, at least 220, at least 2.40, at least 260, at least 280, or at least 300 amino acids. In some embodiments, the RDDP comprises at least 277 amino acids. In some embodiments, the RDDP comprises 200 to 800 amino acids. In some embodiments, the RDDP comprises 277 to 800 amino acids. In some embodiments, the RDDP comprises 300 to 800. 400 to 800, 500 to 800, or 600 to 800 amino acids. In some embodiments, the RDDP comprises 250 to 750, 250 to 700, 250 to 650. 250 to 600, 250 to 550. 250 to 500, 250 to 450, 250 to 400, 250 to 350, 275 to 750,275 to 700, 275 to 650. 275 to 600, 275 to 550, 275 to 500, 275 to 450, 275 to 400, 275 to 350,300 to 750. 300 to 700, 300 to 650. 300 to 600, 300 to 550, 300 to 500, 300 to 450, 300 to 400, or 300 to 350 amino acids.

[0201] In some embodiments, the length of the RDDP is less than 800 amino acids. In some embodiments, the length of the RDDP is less than 800, less than 700, less than 600, less than500, less than 400, less than 300 linked amino acids. In some embodiments, the length of the RDDP is at least 200, at least 220. at least 240, at least 260. at least 280, or at least 300 linked ammo acids. In some embodiments, the length of the RDDP is at least 277 linked amino acids. In some embodiments, the length of the RDDP is 200 to 800 linked ammo acids. In some embodiments, the length of the RDDP is 277 to 800 linked amino acids. In some embodiments, the length of the RDDP is 300 to 800, 400 to 800, 500 to 800, or 600 to 800 linked amino acids. In some embodiments, the length of the RDDP is 250 to 750. 250 to 700, 250 to 650, 250 to 600, 250 to 550, 250 to 500, 250 to 450, 250 to 400, 250 to 350, 275 to 750, 275 to 700, 275 to 650, 275 to 600, 275 to 550, 275 to 500, 275 to 450, 275 to 400, 275 to 350, 300 to 750, 300 to 700, 300 to 650. 300 to 600, 300 to 550, 300 to 500, 300 to 450. 300 to 400, or 300 to 350 linked ammo acids.

[0202] In some embodiments, the RDDP comprises an amino acid sequence that it at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one of SEQ ID NOs:9402-9471. In some embodiments, the RDDP comprises or consists of an amino sequence selected from SEQ ID NOs: 9402-9471. In some embodiments, the present disclosure provides a polynucleotide encoding an RDDP described herein. In some embodiments, the present disclosure provides a polynucleotide encoding an RDDP comprising an amino acid sequence that it at least 90%. 91%. 92%. 93%. 94%, 95%, 96%, 97%, 98%, or 99% identical to one ofSEQ ID NOs: 9402-9471. In some embodiments, the polynucleotide encodes an RDDP comprising or consisting of an amino sequence selected from SEQ ID NOs: 9402-9471. Exemplary RDDP sequences are provided in TABLE 8 below.

[0203] In some embodiments. RDDPs comprise al least 200. at least 225. at least 250. at least 275 at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, at. least 500, at least 525, at least 550, at least 575, at least 600, at least 625, at least 650, at least 675, at least 700, at least 725. at least 750, at least 775 contiguous amino acids of a sequence selected from SEQ ID NOs: 9402-9471.Engineered Proteins

[0204] Any of the nucleic acid sequences that encode the protein sequences disclosed herein may be codon optimized. In some embodiments, effector proteins described herein are encoded by a codon optimized nucleic acid. In some embodiments, LSRs described herein are encoded by a codon optimized nucleic acid. In some embodiments, RDDPs described herein are encoded by a codon optimized nucleic acid In some embodiments, a nucleic acid sequence encoding an effector protein described herein, is codon optimized. This type of optimization can entail a mutation of an effector protein encoding nucleotide sequence to mimic the codon preferences of the intended host organism or cell while encoding the same polypeptide. Thus, the codons can be changed, but the encoded protein remains unchanged. For example, if the intended target cell was a human cell, a human codon-optimized effector protein-encoding nucleotide sequence could be used. As another non-limiting example, if the intended host cell were a mouse cell, then a mouse codon -optimized effector protein-encoding nucleotide sequence could be generated. As another non-limiting example, if the intended host cell were a eukaryotic cell, then a eukaryote codon-optimized effector protein nucleotide sequence could be generated. As another non-limiting example, if the intended host cell were a prokaryotic cell, then a prokaryote codon-optimized effector protein-encoding nucleotide sequence could be generated. Codon usage tables are readily available, for example, at the "Codon Usage Database” available at www.kazusa.or.jp / codon. Accordingly, in some embodiments, effector proteins described herein may be codon optimized for expression in a specific cell, for example,a bacterial cell, a plant cell, a eukaryotic cell, an animal ceil, a mammalian cell, or a human cell. In some embodiments, the effector protein is codon optimized for a human cell.

[0205] It is understood that when describing coding sequences of polypeptides described herein, said coding sequences do not necessarily require a codon encoding a N-terminal Methionine (M) or a Valine (V) as described for the effector proteins described herein. One skilled in the art would understand that a start codon could be replaced or substituted with a start codon that encodes for an amino acid residue sufficient for initiating translation in a host cell. In some embodiments, when a modifying heterologous peptide, such as a fusion partner protein, protein tag or NTS. is located at the N terminus of the effector protein, a start codon for the heterologous peptide serves as a start codon for the effector protein as well. Thus, the natural start codon encoding an amino acid residue sufficient for initiating translation (e.g., Methionine (M) or a Valine (V)) of the effector protein may be removed or absent.

[0206] In some embodiments, LSRs, RDDPs, and / or effector proteins described herein comprise one or more amino acid substitutions as compared to a naturally occurring protein. In some embodiments, the amino acid substitution is a conservative amino acid substitution. In general, a conservative amino acid substitution is the substitution of an amino acid residue for another amino acid residue with similar chemical properties (e.g., size, charge, or polarity). Conservative substitutions may be made by exchanging an amino acid from one of the groups listed below (group 1 to 6) for another amino acid of the same group.

[0207] Amino acid residues may be divided into groups based on common side chain properties, as follows: (group 1) hydrophobic: norieucine (Nle), methionine (Met), Alanine (Ala), Valine (Vai), Leucine (Leu). Isoleucine (lie); (group 2) neutral hydrophilic: Cysteine (Cys), Serine (Ser), Threonine (Thr). Asparagine (Asn), Glutamine (Gin); (group 3) acidic: Aspartic acid (Asp). Glutamic acid (Glu); (group 4) basic: Histidine (His), Lysine (Lys), Arginine (Arg); (group 5) residues that influence chain orientation: Glycine (Gly). Proline (Pro); and (group 6) aromatic: Tryptophan (Trp). Tyrosine ( Ty r). Phenylalanine (Phe). The substitution of one amino acid with another amino acid in a same group listed above may be considered a conservative amino acid substitution. The substitution of one amino acid with another amino acid in a different group listed above may be considered a non-conservative ammo acid substitution.

[0208] In some embodiments, the effector protein is an engineered effector protein and comprises an ammo acid sequence that is at least 90%, at least 95%, at least 97%. at least 98%, or at least 99% identical to SEQ ID NO: 9401, wherein the polypeptide comprises at least oneamino acid substitution relative to SEQ ID NO: 9401, wherein the amino acid substitution is at aposition selected from K58, 180, T84, KI 05, N193, C202, S209, G210, A218, D220, E225, C246, N286, M295, M298, A306, Y315, Q360, and a combination thereof. In some embodiments, the poly peptide comprises an amino acid sequence that is 1 ( )( )% identical to SEQ ID NO: 9401, with the exception of at least one amino acid substitution relative to SEQ ID NO: 9401, wherein the ammo acid substitution is a position selected from K58, 180, 1'84, KI 05, N193, C202, S209, G210, A218, D220, E225, C246, N286, M295, M298, A306, Y315, Q360, and a combination thereof. In some embodiments, the amino acid substitution is selected from K58X, 180X, T84X, K105X. N193X. C202X, S209X. G210X. A218X, D220X, E225X, C246X. N286X, M295X, M298X. A306X, Y315X. and Q360X. wherein X is selected fromR, K, and H.

[0209] In some embodiments, the effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%. at least 97%. at least 98%, or at least 99% identical to SEQ ID NO: 9401, wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 9401, wherein the amino acid substitution is selected from I80R, T84R, K105R, C202R, G210R. A218R, D220R, E225R, C246R, Q360R, I80K, T84K. G210K, N193K, C202K. A218K, D220K, E225K, C246K, N286K A306K, Q360K, I80H, T84H, K105H, G210H. C202H, A218H. D220H. E225H, C246H, Q360H. K58W, S209F, M295W, M298L, Y315M, D220R / A306K and D220R / K250N and a combination thereof. In some embodiments, the polypeptide comprises an amino acid sequence that is 100% identical to SEQ ID NO: 9401. with the exception of at least one ammo acid substitution relative to SEQ ID NO: 9401, wherein the amino acid substitution is selected from T80R. T84R, K105R, C202R, G210R, A218R, D220R, E225R, C246R, Q360R, I80K, T84K, G210K, N193K, C202K, A2I8K, D220K, E225K, C246K, N286K, A306K, Q360K, I80H, T84H, K105II, G210H, C202H, A218H, D220H, E22.5H. C246H, Q360H, K58W, S209F,M295W. M298L, Y315M. D220R / A306K and D220R / K250N and a combination thereof. In some embodiments, these engineered effector proteins demonstrate enhanced nuclease activity' relative to the wild-type effector protein.

[0210] In some embodiments, the effector protein is an engineered effector protein and comprises an ammo acid sequence that is at least 90%, at least 95%. at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 9401, wherein the polypeptide comprises at least one ammo acid substitution relative to SEQ ID NO: 9401, wherein the amino acid substitution is selected from D237A, D418A, D418N. E335A, and E335Q, and a combination thereof Insome embodiments, the polypeptide comprises an amino add sequence that is 100% identical to SEQ ID NO: 9401, with the exception of at least one amino acid substitution relative to SEQ ID NO: 9401, wherein the amino acid substitution is selected from D237A. D418A, D418N, E335A. and E335Q, and a combination thereof. In some embodiments, these engineered effector proteins demonstrate reduced or abolished nuclease activity relative to the wild-type effector protein. TABLE 5 provides the exemplary ammo acid alterations relative to SEQ ID NO: 9401 useful in compositions, systems, and methods described herein.

[0211] As used herein, the term “catalytically inactive effector protein," also referred to as a "‘dCas” protein, as used herein, refers to an effector protein that is modified relative to a naturally-occurring effector protein to have a reduced or eliminated catalytic activity7relative to that of the naturally-occurring effector protein, but retains its ability to interact with a guide nucleic acid. The catalytic activity that is reduced or eliminated is often a nuclease activity. The naturally-occurring effector protein may be a wildtype protein. In some embodiments, the catalytically inactive effector protein is referred to as a catalytically inactive variant of an effector protein, e.g., a Cas effector protein. In some embodiments, the catalytically inactive effector protein is referred to as a dead Cas protein or a dCas protein ,

[0212] In some embodiments, the effector protein is an engineered effector protein and comprises an ammo acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%. or at least 99% identical to SEQ ID NO: 9278, wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 9278, wherein the amino acid substitution is at a position selected from 12, T5, KI 5, Rl 8, H20, S21, L26, N30, E33, E34. A35, K37, K38, R41, N43, Q54, Q79R, K92E. K99R, S 108, E1 Q9, HHO. Gi l l , D I 13, Tl 14, P H6. K i i8. El 19, A12I , N 132. K135, Q138, V I 39, N148, LI 49, E157, El 64, El 66, El 70, Y180, LI 82,Q183, KI 84, SI 86, KI 89, S196, SI 98, K200, 1203, S205, K206, Y207, H208, N209, Y220, S223, E258, K281, K348, N355, S362, N406, K43.5, 1471, 1489, Y490. F491, D495, K496,K498, K500. D50I, V502, K.504, S505, D506, V521, N568, S579, Q612, S638. F701, P707, and a combination thereof. In some embodiments, the polypeptide comprises an ammo acid sequence that is 100% identical to SEQ ID NO: 9278, with the exception of at least one amino acid substitution relative to SEQ ID NO: 9278, wherein the amino acid substitution is at a position selected from 12, T5, KI 5, Rl 8. H20, S21 , L26, N30, E33, E34, A35, K37. K38. R41,N43, Q54. Q79R, K92E, K99R, SI08, E109. H1 I0, Gi l l. D113, TH4, Pl 16, KI 18, E119. A121 , N132, K135, Q138. V139, N148, L149, El 57, El 64, El 66, El 70, Y180, Li 82, Q183, K I 84. S186, KI 89, S196, S 198, K200, 1203, S205, K206, Y207, H208. N209, Y220, S223,E258, K281, K348, N355, S362, N406, K435, 1471 , 1489, Y490, F491, D495, K496, K498, K500, D501 , V502, K504, S505, D506, V521 , N568, S579, Q612, S638, F701, P707, and a combination thereof. In some embodiments, the amino acid substitution is selected from I2X, T5X. K15X, R18X, H20X, S21X, L26X. N30X, E33X, E34X, A35.X, K37X. K38X. R41X, N43X, Q54X, Q79RX, K92EX, K99RX, S108X, E109X, H110X, Gi l IX, D113X, T1 I4X.P116X. K118X, E119X, A121X, N132X, K135X, Q138X, V139X, N148X, L149X, E157X, E164X, E166X, E170X, Y180X, L182X, Q183X, K184X, S186X, K189X, S196X, S198X, K20QX, I203X, S205X, K206X, Y207X, H208X, N209X, Y220X, S223X, E258X, K281X, K348X, N355X. S362X. N406X. K435X, 1471X, I489X, Y490X. F491 X, D495X, K496X. K498X, K500X, D50LX, V502X, K504X. S505X, D506X. V521X, N568X, S579X, Q612X,S638X, F701X, P707X, wherein X is selected from R, K, and H.

[0213] In some embodiments, the effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%. at least 98%, or at least 99% identical to SEQ ID NO: 9278, wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 9278, wherein the amino acid substitution is selected from T5R, L26R, L26K, A121Q, V139R, S198R, S223P, E258K, 147 IT, S579R, F701R. P707R, K189P, S638K, Q54R, Q79R, Y220S. N406K, El 19S, K92E. K435Q, N568D, and V521T, and a combination thereof. In some embodiments, the polypeptide comprises an amino acid sequence tha t is 100% identical to SEQ ID NO: 9278, with the exception of at least one amino acid substitution relative to SEQ ID NO: 9278, wherein the amino acid substitution is selected from T5R, L26R, L26K, A121Q, V139R, S198R, S223P, E258K, 147 IT, S579R, F701R. P707R, KI 89P, S638K, Q54R, Q79R, Y220S, N406K, El 19S, K92E, K435Q, N568D, and V521 T, and a combination thereof. In some aspects, these engineered effector proteins demonstrate enhanced nuclease activity relative to the w i Id -ty pe effector protein

[0214] In some embodiments, the effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%. at least 98%, or at least 99% identical to SEQ ID NO: 9278. wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 9278, wherein the amino acid substitution is selected from L26K / A121Q, L26R / A121Q, K99R / L149R, K99R / N148R, L 149R / H208R, S362R / L26R L26R / N148R, L26R / H208R. N30R / N 148R, L26R / K99R, L26R / P707R,L26R / L149R, L26R / N30R, L26R / N355R, L26R / K28IR. L26R / S108R, L26R / K348R. T5R / V139R, I2R / V139R, K99R / S I 86R. L26R / A673G, L26R / Q674R, S579R / L26K, F701R / E258K, T5R / L26K. L26R / K435Q, L26R / G685R, L26R / Q674K, L26R / P699R,L26R / T70E, L26R / Q232R, L26R / T252R, L26R / P679R, L26R / E83K, L26R / E73P, L26R / K248E, L26R, T5R7 S223P, S579R / S223P, L26R / S223P, T5R / Al 21 Q, L26R / A696R, S198R / 147 IT, L26R / N153R, L26R / E682R, L26R / D703R, Q612R' L26K, L26R / 147 IT, K348R / L26K, S579R / I471T, L26R / V228R, T5R / S638K, S579R / K189P, S579R / E258K, L26RZK260R, L26R / S638K, S579R / Y220S, T5R / 1471 T. L26R / F233R. L26R / V521T,F701R / A121Q, L26R / G361R, S198R / E258K, L26R / S472R, T5R / Y220S, L26R / A150K, L26R / S684R, L26R / E157R, L26R / K248R, F701R / L26K, S198R / N406K, S198R / Y220S, SI98R / S638K, S198R / V521T, S579R / A121 Q, K.348R / Y220S. S198R / K.189P, L26R / E242R, L26R / K678R, T5R / N406K, L26R / 1158K, T5R / V521T, L26R / N259R, L26R / K257R. L26R / K256R, T5R / K.189P. L26R / C405R, S579R / V521T, S579R / N406K, T5R / K92E,T5R / E258K, L26R / I97R, S579R / S638K, T5R / K435Q, F701R / S638K, L26R / L236R, F701R / I471T, Q612R / S223P, F701R / S223P, S198R / E1 19S, S579R / K92E, L26R / E715R, Q612R / I471T, F701R / Y220S, S198R / S223P, and L26R / K266R, and a combination thereof. In some embodiments, the polypeptide comprises an amino acid sequence that is 100% identical to SEQ ID NO: 9278, with the exception of at least one amino acid substitution relative to SEQID NO: 9278, wherein the amino acid substitution is selected from L26K / A121Q, L26R'A12IQ, K99R / L149R, K99R / N148R. L149R / H208R. S362R''L26R L26R / N148R, L26R / H208R, N30R / N148R, L26R / K99R, L26R / P7O7R. L26R'L149R. L26R / N30R.L26R / N355R, L26R / K281R. L26R / SI08R, L26R / K348R, T5R / VI39R, I2R / V139R, K99R / S186R, L26R / A673G, L26R / Q674R, S579R / L26K, F701R / E258K, T5R / L26K,L26R / K435Q, L26R / G685R, L26R / Q674K, L26R / P699R, L26R / T70E, L26R / Q232R,L26R / T252R, L26R / P679R, L26R / E83K, L26R / E73P, L26R / K248E, L26R, T5R / S223P, S579R7 S223P, L26R7 S223P, T5R / A 12 I Q, L26R / A696R. SI 98R / 147 IT, L26R / NI53R, L26R / E682R, L26R / D703R, Q612R / L26K, L26R / 147 IT, K348R / L26K, S579R / I471T, L26R / V228R, T5R / S638K, S579R / K189P, S579R / E258K, L26R / K260R, L26R / S638K,S579R / Y220S, T5R / 1471T. L26R / F233R, L26R / V521T, F70IR / A121Q, L26R / G36IR, S198R / E258K, L26R / S472R, T5R / Y220S, L26R / A150K, L26R / S684R, L26R / E157R, L26R / K248R, F701R / L26K, SI98R / N406K, S198R / Y220S, S198R / S638K, S198R / V521T, S579RZA121Q, K348R / Y220S, S198R / K189P, L26R / E242R, L26R / K678R, T5R / N406K, L26R / I158K, T5R / V521T. L26R / N259R, L26R / K257R, L26R / K256R, T5R / KI 89P,L26R / C405R. S579R / V521T, S579R / N406K. T5R / K92E, T5R / E258K, L26R / I97R, S579R / S638K, T5R / K435Q, F701R / S638K, L26R / L236R, F701R / I471T, Q612R / S223P, F701R / S223P, S198R / E119S, S579R / K92E, L26R / E715R, Q612R / I471T, F70IR / Y220S,S 198R / S223P, and L26R / K266R, and a combination thereof. In some aspects, these engineered effector proteins demonstrate enhanced nuclease activity relative to the wild-type effector protein.

[0215] In some embodiments, the effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 9278. wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 9278 wherein the amino acid substitution is selected from E157A, El 64 A, E164L. E166A, El 661. E170A, I489A, I489S. Y490S, Y490A, F491 A. F491S, F491G, D495G, D495R, D495K, K496A. K496S, K498A. K498S, K500A. K500S, D50IR, D501G, D501K, V502A, V502S, K504A, K504S, S505R, D506A. and a combination thereof. In some embodiments, the polypeptide comprises an amino acid sequence that is 100% identical to SEQ ID NO: 9278. with the exception of at least one ammo acid substitution relative to SEQ ID NO: 9278, wherein the amino acid substitution is selected from EI 57A, E164A, E164L, E166A. El 661, E170A, I489A, I489S, Y490S. Y490A. F491A, F491S, F49IG. D495G, D495R, D495K, K496A, K496S, K498A, K498S, K500A, K500S,D501R, D501G, D501K. V502A, V502S, K504A, K504S, S505R, D506A, and a combmation thereof. In some embodiments, these engineered effector proteins comprise a nickase acti vity .

[0216] In some embodiments, the effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 9278, wherein amino acids S478-S505 have been deleted, in some embodiments, the effector protein is an engineered effector protein that is at least 90%, at least 95%, at least 97%. at least 98%. or at least 99% identical to SEQ ID NO: 9278. wherein amino acids S478-S505 have been deleted and replaced with SDLYIERGGDPRDVHQQVETKl’KGKRKSEIRILKIR (SEQ ID NO: 9698) or SDYIVDHGGDPEKVFFETKSKKDKTKRYKRR (SEQ ID NO: 9699) In some embodiments, the effector protein is an engineered effector protein and comprises an ammo acid sequence that is at least 90%, at least 95%. at least 97%, at least 98%, at least 99% identical, or is 100% identical to SEQ ID NO: 9700. In some embodiments, the effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical, or is 100% identical to SEQ ID NO: 9701.

[0217] In some embodiments, the effector protein is an engineered effector protein and comprises an ammo acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%,or at least 99% identical to SEQ ID NO: 9278, wherein the polypeptide comprises at least one ammo acid substitution relative to SEQ ID NO: 9278 wherein the amino acid substitution is selected from D369A, D369N, D658A, D658N, E567A, E567Q, and a combination thereof. In some embodiments, the polypeptide comprises an ammo acid sequence that is 100% identical to SEQ ID NO: 9278, with the exception of at least one ammo acid substitution relative to SEQ ID NO: 9278, wherein the amino acid substitution is selected from D369A, D369N. D658A, D658N, E567A, E567Q, and a combination thereof. In some aspects, these engineered effector proteins demonstrate reduced or abolished nuclease activity relative to the wild-type effector protein TABLE 6 provides the exemplary amino acid alterations relative to SEQ ID NO: 9278 useful in compositions, systems, and methods described herein.

[0218] In some embodiments, the RDDP is an engineered RDDP and comprises an amino acid sequence that it at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one of SEQ ID NOs: 9472-9482. In some embodiments, the RDDP comprises or consists of an amino sequence selected from SEQ ID NOs: 9472-9482.

[0219] In some embodiments, the present disclosure provides a polynucleotide encoding anRDDP described herein In some embodiments, the present disclosure provides a polynucleotide encoding an RDDP comprising an amino acid sequence that it at least 90%, 91 %. 92%. 93%, 94%, 95%. 96%. 97%, 98%, or 99% identical to one of SEQ ID NOs: 9472- 9482. In some embodiments, the polynucleotide encodes an RDDP comprising or consisting of an amino sequence selected from SEQ ID NOs: 9472-9482,

[0220] In some embodiments, RDDPs comprise at least 200, at least 225, at least 250, at least 275 at least 300, at least 325, at least 350, at least 375. at least 400, at least 425. at least 450, at least 475, at least 500, at least 525, at least 550, at least 575, at least 600, at least 625, at least 650, at least 675, at least 700, at least 725, at least 750, at least 775 contiguous amino acids of a sequence selected from SEQ ID NOs: 9472-9482,

[0221] Exemplary' engineered RDDPs are provided in TABLE 9 below. For each engineered RDDP, the parental RDDP is listed followed by the ammo acid mutations in parentheses. For example, 2691319 (D12R-D72R-N195R) refers to an engineered RDDP based on 2691319 (SEQ ID NO: 9413) and comprising the mutations D12R, D72R, and N195R.

[0222] Engineered effector proteins may provide enhanced catalytic activity (e.g., nuclease or nickase activity) as compared to a naturally occurring nuclease or nickase. Engineered effector proteins may provide enhance nucleic acid binding activity, e.g, enhanced binding of a guide nucleic acid and / or target nucleic acid, and / or may demonstrate a stronger affinity for a targetnucleic acid sequence. Without wishing to be bound by theory, substitution of positively charged amino acids is thought to increase the interaction between the effector proteins and / or RDDPs and the negatively charged target nucleic acid sequences. By way of non-limiting example, some engineered proteins exhibit optimal activity at lower salinity and viscosity than the protoplasm of their bacterial cell of origin. Also, by way of non-limiting example, bacteria often comprise protoplasmic salt concentrations greater than 250 mM and room temperature intracellular viscosities above 2 centipoise, whereas engineered proteins exhibit optimal activity (e.g., cis-cleavage activity) at salt concentrations below 150 rnM and viscosities below 1.5 centipoise. The present disclosure leverages these dependencies by providing engineered proteins in solutions optimized for their activity and stability.

[0223] Compositions and systems described herein may comprise an engineered effector protein and / or RDDP in a sol ution comprising a room temperature viscosity of less than about 15 centipoise, less than about 12. centipoise, less than about 10 centipoise, less than about 8 centipoise, less than about 6 centipoise, less than about 5 centipoise, less than about 4 centipoise, less than about 3 centipoise, less than about 2 centipoise, or less than about 1.5 centipoise.

[0224] Compositions and systems may comprise an engineered effector protein anchor RDDP in a solution comprising an ionic strength of less than about 500 mM. less than about 400 mM, less than about 300 mM, less than about 250 mM, less than about 200 mM, less than about 150 mM, less than about 100 mM, less than about 80 mM, less than about 60 mM, or less than about 50 mM. Compositions and systems may comprise an engineered effector protein and / or RDDP and an assay excipient, which may stabilize a reagent or product prevent aggregation or precipitation, or enhance or stabilize adetectable signal (e g., a fluorescent signal). Examples of assay excipients include, but are not limited to, saccharides and saccharide derivatives (e.g., sodium carboxy methyl cellulose and cellulose acetate), detergents, glycols, polyols, esters, buffering agents, alginic acid, and organic solvents (e.g , DMSO).

[0225] An engineered protein may comprise a modified form of a wild type counterpart protein. The modified form of the wild type counterpart may comprise an ammo acid change (e.g., deletion, insertion, or substitution) that reduces the nucleic acid-cleaving activity of the effector protein relative to the wild type counterpart. For example, a nuclease domain (e.g.,RuvC domain) of an effector protein may be mutated relative to a wild type counterpart effector protein so that it is comprises reduced nuclease activity. The modified form of the effector protein may comprise increased nickase activity relative to that of a wild-type counterpart.Modulation of Effector protein activity

[0226] The effector proteins of the present disclosure may have nuclease activity or nickase activity' on target nucleic acids. In some embodiments the effector protein of the present disclosure has a combination of the above activities on a target nucleic acid. In some embodiments the cleavage activity of the effector proteins is modulated between nuclease activity7and nickase activity- on the target nucleic acid.

[0227] In some embodiments the cleavage activity of the effector protein on the target nucleic acid is modulated based on the length of the spacer sequence of a guide nucleic acid. In some embodiments the spacer length confers nuclease activity to the effector protein on the target nucleic acid. In some embodiments the spacer length confers nickase activity' to the effector protein on the target nucleic acid. In some embodiments, the spacer length is 10-20 nucleotides. In some embodiments, the spacer length is 11-17 nucleotides. In some embodiments, the spacer length is 14-16 nucleotides. In some embodiments, the spacer length is 10, 12, 13, 14, 15. 16 or 17 nucleotides In some embodiments, the spacer length is 15 nucleotides. Fusion proteins

[0228] In some embodiments, the present disclosure provides a fusion protein comprising an effector protein, an RDDP. an I SR. or a combination thereof. In some embodiments, the present disclosure provides a fusion protein comprising an effector protein, a base editing enzyme, an LSR, or a combination thereof. In some embodiments, the fusion protein comprises, from N-terminus to C -terminus:(a) an effector protein, an RDDP, and an LSR;(b) an effector protein, an LSR, and an RDDP;(c) an RDDP, an effector protein, and an LSR;(d) an RDDP, an LSR, and an effector protein; (e) an LSR. an effector protein, and an RDDP;(f) an LSR, an RDDP, and an effector protein;(g) an effector protein, a base editing enzyme, and an LSR;(h) an effector protein, an LSR, and a base editing enzyme;(i) a base editing enzyme, an effector protein, and an LSR; (j) a base editing enzyme, an LSR, and an effector protein;(k) an LSR, an effector protein, and a base editing enzyme;0) an LSR. a base editing enzyme, and an effector protein;(m) an effector protein and an LSR; or(n) an LSR and an effector protein.

[0229] In some embodiments, the present disclosure provides a fusion protein comprising an effector protein and an LSR, wherein the effector protein lacks nuclease activity. Exemplary embodiments of effector proteins that lack nuclease activity are provided in TABLE 5 and TABLE 6. In such embodiments, the effector protein directs the activity of the LSR to a target nucleic acid sequence in combination with a guide RNA.Synthesis, Isolation and Assaying

[0230] LSRs. RDDPs, effector proteins and fusion proteins of the present disclosure of the present disclosure may be synthesized, using any suitable method. Additionally, nucleic acids, including mRNA encoding LSRs, RDDPs, effector proteins, and fusion proteins of the present disclosure may be synthesized using suitable methods. LSRs, RDDPs, effector proteins, and fusion proteins of the present disclosure may be produced in vitro or by eukaiyotic cells or by prokaryotic cells. LSRs, RDDPs, effector proteins, and fusion proteins can be further processed by unfolding, e.g. heat denaturation, dithiothreitol reduction, etc. and may be further refolded, using any suitable method.

[0231] Methods of generating and assaying the LSRs, RDDPs. effector proteins, and fusion proteins described herein are well known to one of skill in the art Examples of such methods are described in the Examples provided herein Any of a variety of methods can be used to generate an effector protein disclosed herein. Such methods include, but are not limited to, site-directed mutagenesis, random mutagenesis, combinatorial libraries, and other mutagenesis methods described herein (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999); Gillman et al.. Directed Evolution Library Creation: Methods and Protocols (Methods in Molecular Biology) Springer. 2nd ed (2014)). One non-limiting example of a method for preparing an effector protein is to express recombinant nucleic acids encoding the effector protein in a suitable microbial organism, such as a bacterial cell, a yeast cell, or other suitable cell, using methods well known in the art.

[0232] In some embodiments, an LSR, RDDP, base editing enzyme, effector protein, and / or fusion protein provided herein is an isolated protein. In some embodiments, an LSR, RDDP, effector protein, base editing enzyme, and / or fusion protein described herein can be isolated and purified for use in compositions, sy stems. and / or methods described herein. Methods described here can include the step of isolating LSRs, RDDPs. effector proteins, and / or fusion proteins described herein. An isolated LSR, RDDP, effector protein, base editing enzyme, and / or fusion protein provided herein can be isolated by a variety of methods well-known in the art, for example, recombinant expression systems, precipitation, gel filtration, ionexchange, reverse-phase and affinity chromatography, and the like. Other well-known methods are described in Deutscher et al.. Guide to Protein Purification: Methods inEnzymology, Vol. 182, (Academic Press, (1990)). Alternatively, the isolated polypeptides of the present disclosure can be obtained using well-known recombinant methods (see, e.g., Sambrook et al.. Molecular Cloning: A Laboratory' Manual. Third Ed., Cold Spring Harbor Laboratory7, New York (2001 ); and Ausubel et al.. Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD ( 1999)). The methods and conditions for biochemical purification of a polypeptide described herein can be chosen by those skilled in the art, and purification monitored, for example, by a functional assay.

[0233] LSRs. RDDPs, effector proteins, and fusion proteins disclosed herein may be covalently linked or attached to a tag, e g., a purification tag A purification tag, as used herein, can be an amino acid sequence which can attach or bind with high affinity to a separation substrate and assist in isolating the protein of interest from its environment, which can be its biological source, such as a cell lysate Attachment of the purification tag can be at the N or C terminus of the LSRs, RDDPs, effector proteins, and / or fusion proteins. Furthermore, an amino acid sequence recognized by a protease or a nucleic acid encoding for an amino acid sequence recognized by a protease, such as TEV protease or the HRV3C protease can be inserted between the purification tag and the LSR, RDDP, effector protein, base editing enzyme, and / or fusion protein, such that biochemical cleavage of the sequence with the protease after initial purification liberates the purification tag. Purification and / or isolation can be through high performance liquid chromatography (HPLC), exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. Examples of purification tags are as described herein.Guide Nucleic A elds

[0234] The compositions, systems, and methods of the present disclosure may comprise a guide nucleic acid or a use thereof. Unless otherwise indicated, compositions, systems and methods comprising guide nucleic acids or uses thereof, as described herein and throughout, include DNA molecules, such as expression vectors, that encode a guide nucleic acid.Accordingly, compositions, systems, and methods of the present disclosure comprise a guide nucleic acid or a nucleotide sequence encoding the guide nucleic acid.

[0235] In general, a guide nucleic acid is a nucleic acid molecule, at least a portion of which may be bound by an effector protein, thereby forming a ribonucleoprotein complex (RNP). Another portion of the guide nucleic acid molecule can comprise a spacer region which is complementary to at least a portion of the target nucleic acid sequence. In some embodiments, the guide nucleic acid imparts activity or sequence selectivity to the effector protein. When complexed with an effector protein, guide nucleic acids can bring the effector protein into proximity of a target nucleic acid. The guide nucleic acid spacer region may hybridize to a target nucleic acid or a portion thereof. In some embodiments, when a guide nucleic acid and an effector protein form an RNP, at least a portion of the RNP binds spacer region, recognizes, and / or hybridizes to a target nucleic acid. Those skilled m the art in reading the below specific examples of guide nucleic acids as used in RNPs described herein, will understand that in some embodiments, a RNP can hybridize, via the spacer region, to one or more target sequences in a target nucleic acid, thereby allowing the RNP to modify and / or recognize a target nucleic acid or sequence contained therein.

[0236] A guide nucleic acid, as well as any components thereof (e g., spacer region, repeat region, linker) may comprise one or more deoxyribonucleotides, ribonucleotides, biochemically or chemically modified nucleotides (e.g., one or more sequence modifications as described herein), and any combinations thereof. A guide nucleic acid may comprise a naturally occurring sequence. A guide nucleic acid may comprise a non-naturally occurring sequence, wherein the sequence of the guide nucleic acid, or any portion thereof, may be different from the sequence of a naturally occurring nucleic acid. The guide nucleic acid may be chemically synthesized or recombinantly produced. Guide nucleic acids and portions thereof may be found in or identified from a CRISPR array present in the genome of a host organism or cell.

[0237] Guide nucleic acids, while often being referred to as a guide RNA, may include deoxyribonucleosides, ribonucleosides, chemically modified nucleosides, or any combination thereof.

[0238] In some embodiments, the guide nucleic acid comprises a hairpin or stem-loop structure that is recognized by the effector protein. The hairpin or stem-loop structure may comprise a repeat region. In some embodiments, the guide nucleic acid comprises at least a portion of a tracrRNA sequence. In nature, a tracrRNA is a guide nucleic acid that has trans activating activity on a target nucleic acid through a repeat hybridization sequence. In some embodiments, guide nucleic acids of the instant di sclosure do not comprise a repeat hybridization sequence. In some embodiments, guide nucleic acids of the instant disclosure do not comprise a tracrRNA. In some embodiments, e.g., systems comprising CasPhi proteins and fusions thereof, the guide nucleic acid does not comprise a tracrRNA sequence but comprises a repeat sequence to which the CasPhi protein binds.

[0239] In some embodiments, the guide nucleic acid comprises a repeat region that interacts with the effector protein The term, “repeat region” may be used interchangeably herein with the term, “repeat sequence.’’ In some embodiments, an effector protein interacts with a repeat region. In some embodiments, an effector protein does not interact with a repeat region. Typically, the repeat region is adjacent to the spacer region. In certain embodiments, the repeat region is followed by the spacer region in the 5’ to 3’ direction. Exemplary’ repeat region sequences for exemplary effector proteins provided herein are shown in TABLE 4.Spacer Sequences

[0240] In general, guide nucleic acids comprise a spacer sequence that hybridizes with a target sequence of a target nucleic acid. The term, “spacer sequence,” refers to a region of the guide nucleic acid that hybridizes to a target sequence of a target nucleic acid. The terms “spacer sequence” and “spacer region” are used interchangeably herein and throughout. Tire spacer sequence may comprise a sequence that is complementary with a target sequence of a target nucleic acid. In some embodiments, the spacer sequence is complementary to the target sequence on the target strand of a dsDNA molecule. In some embodiments, the spacer sequence is complementary' to the target sequence on the non-target strand of a dsDNA molecule. The spacer sequence can function to direct the guide nucleic acid to the target nucleic acid for detection and / or modification of the target nucleic acid. The spacer sequence may’ be complementary' to a target sequence that is adjacent to a PAM that is recognizable by an effector protein of interest.

[0241] In some embodiments, the spacer sequence is 15-28 linked nucleotides in length. In some embodiments, the spacer sequence is 15-26, 15-24. 15-22, 15-20, 15-18, 16-28, 16-26, 16-24, 16-22, 16-20, 16-18, 17-26, 17-24, 17-22, 17-20, 17-18, 18-26, 18-24, or 18-22 linked nucleotides in length. In some embodiments, the spacer sequence is 18-24 linked nucleotides in length. In some embodiments, the spacer sequence is at least 15 linked nucleotides in length.In some embodiments, the spacer sequence is at least 16, 18, 20, or 22 linked nucleotides in length. In some embodiments, the spacer sequence comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29. or 30 nucleotides. In some embodiments, the spacer sequence is at least 17 linked nucleotides in length. In some embodiments, the spacer sequence is at least 18 linked nucleotides in length. In some embodiments, the spacer region is at least 20 linked nucleotides in length. In some embodiments, the spacer sequence is at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to a target sequence of the target nucleic acid. In some embodiments, the spacer sequence is 100% complementary' to the target sequence of the target nucleic acid. In some embodiments, the spacer sequence comprises at least 15 contiguous nucleotides that are complementary' to the target nucleic acid.It is understood that the sequence of a spacer sequence need not be 100% complementary' to that of a target sequence of a target nucleic acid to hybridize or hybridize specifically to the target sequence. The spacer sequence may comprise at least one nucleotide that is not complementary' to the corresponding nucleotide of the target sequence. In some embodiments the spacer sequence is less than 100% complementary' to the target sequence, but still can bind to the target sequence. In some embodiments the spacer sequence is 90%, 91%, 92%, 93%, 94%. 95%. 96%, 97%, 98%, 99%, or 100% complementary to the target sequence. In some embodiments, the target sequence is selected from SEQ ID NO: 8965-9266.10242] In some embodiments, a guide nucleic acid, a spacer region thereof or a spacer sequence thereof, comprises at least 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21. 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides that are complementary'' to a eukaryotic sequence. Such a eukaryotic sequence is a sequence of nucleotides that is present in a host eukaryotic cell. Such a sequence of nucleotides i s distinguished from nucleotide sequences present in other host cells, such as prokaryotic cells, or viruses. By way of non-limiting example, said sequences present in a eukaryotic cell can be located in a gene, an exon, an intron, or a non-coding (e.g., promoter or enhancer) region. In some embodiments, a linker is present between the spacer and repeat sequences.

[0243] In some embodiments, a guide nucleic acid comprises a spacer length that confers nickase activity to a Cas enzyme that otherwise comprises nuclease activity when used with guide nucleic acids having a different spacer length. In some embodiments, the Cas enzyme is a Type V Cas enzyme. In some embodiments, the Cas enzyme is a CasPhi enzyme. In some cases, the Cas enzyme is CasPhi. 12 (SEQ ID NO: 9278). In some cases, the spacer length that confers nickase activity is 14-16 nucleotides. In some cases, the spacer length is 15 nucleotides.Nucleic acid linkers

[0244] In some embodiments, a guide nucleic acid for use with compositions, systems, and methods described herein comprises one or more linkers, or a nucleic acid encoding one or more linkers. In some embodiments, the guide nucleic acid comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten linkers. In some embodiments, the guide nucleic acid comprises one, two, three, four, five, six, seven, eight, nine, or ten linkers. In some embodiments, the guide nucleic acid comprises two or more linkers In some embodiments, at least two or more linkers are the same. In some embodiments, at least two or more linkers are not same.

[0245] In some embodiments, a linker comprises one to ten, one to seven, one to five, one to three, two to ten. two to eight, two to six. two to four, three to ten. three to seven, three to five, four to ten, four to eight, four to six, five to ten, five to seven, six to ten, six to eight, seven to ten, or eight to ten linked nucleotides. In some embodiments, the linker comprises one. two, three, four, five, six, seven, eight, nine, or ten linked nucleotides. In some embodiments, a linker comprises a nucleotide sequence of 5‘-GAAA-3‘ (SEQ ID NO: 9695).|0246] In some embodiments, a guide nucleic acid comprises one or more linkers connecting one or more repeat sequences In some embodiments, the guide nucleic acid comprises one or more linkers connecting one or more repeat sequences and one or more spacer sequences. In some embodiments, the guide nucleic acid comprises at least two repeat sequences connected by a linker.Repeat Sequences

[0247] Guide nucleic acids described herein may comprise one or more repeat sequences. In some embodiments, a repeat sequence comprises a nucleotide sequence that is not complementary to a target sequence of a target nucleic acid. In some embodiments, a repeat sequence comprises a nucleotide sequence that may interact with an effector protein In some embodiments, a repeat sequence includes a nucleotide sequence that is capable of forming aguide nucleic acid-effector protein complex (e.g., a RNP complex). Tn some embodiments, the repeat sequence may also be referred to as a “protein-binding segment.’’

[0248] In some embodiments, a repeat sequence is adjacent to a spacer sequence. In some embodiments, a repeat sequence is followed by a spacer sequence in the 5’ to 3’ direction. In some embodiments, a repeat sequence is adjacent to an intermediary sequence. In some embodiments, a repeat sequence is 3‘ to an intermediary sequence. In some embodiments, an intermediary sequence is followed by a repeat sequence, which is followed by a spacer sequence in the 5’ to 3’ direction. In some embodiments, a repeat sequence is linked to a spacer sequence and / or an intermediary sequence. In some embodiments, a guide nucleic acid comprises a repeat sequence linked to a spacer sequence, which may be a direct link or by any suitable linker, examples of which are described herein.

[0249] In some embodiments, the spacer sequence(s) and the repeat sequence(s) of the guide nucleic acid are present within the same polynucleotide molecule. In some embodiments, a spacer sequence is adjacent to a repeat sequence. In some embodiments, a spacer sequence follows a repeat sequence in a 5’ to 3’ direction. Tn some embodiments, a spacer sequence precedes a repeat sequence in a 5’ to 3’ direction. In some embodiments, the spacer(s) and repeat sequence(s) are linked directly to one another. In some embodiments, a linker is present between the spacer(s) and repeat sequence(s). Linkers may be any suitable linker. In some embodiments, the spacer sequence(s) and the repeat sequence(s) of the guide nucleic acid are present in separate polynucleotide molecules, which are joined to one another by base pairing interactions.

[0250] In some embodiments, the repeat sequence comprises two sequences that are complementary to each other and hybridize to form a double stranded RNA duplex (dsRNA duplex). In some embodiments, the two sequences are not directly linked and hybridize to form a stem loop structure. In some embodiments, the dsRNA duplex comprises 5, 10, 15, 20 or 25 base pairs (bp) In some embodiments, not all nucleotides of the dsRNA duplex are paired, and therefore the duplex forming sequence may include a bulge. In some embodiments, the repeat sequence comprises a hairpin or stem-loop structure, optionally at the 5’ portion of the repeat sequence. In some embodiments, a strand of the stem portion comprises a sequence and the other strand of the stem portion comprises a sequence that is, at least partially, complementary.In some embodiments, such sequences may have 65% to 100% complementarity (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementarity). In some embodiments, a guide nucleic acid comprises nucleotide sequence that when involved in hybridization events mayhybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g. , a bulge, a loop structure or hairpin structure, etc.).

[0251] Exemplary repeat sequences are provided in TABLE 4.Intermediary Sequence

[0252] Guide nucleic acids described herein may comprise one or more intermediary sequences. Tn general, an intermediary7sequence used in the present disclosure is not transactivated or transactivating. An intermediary sequence may also be referred to as an intermediary RNA, although it may comprise deoxyribonucleotides instead of or in addition to ribonucleotides, and / or modified bases. In general, the intermediary' sequence non-covalently binds to an effector protein. In some embodiments, the intermediary sequence forms a secondary structure, for example in a cell, and an effector protein binds the secondary structure.

[0253] In some embodiments, an intermediary RNA is a nucleotide sequence in a handle sequence, wherein the intermediary RNA sequence is capable of, at least partially, being non- covalently bound to an effector protein to form a complex (e.g. , an RNP complex). In general, an intermediary RNA sequence is not a transactivating nucleic acid in systems, methods, and compositions described herein.[02541 In some embodiments, a length of the intermediary' sequence is at least 30. 50, 70, 90, 1 10. 130, 150, 170, 190. or 210 linked nucleotides. In some embodiments, a length of the intermediary sequence is not greater than 30, 50, 70, 90, 1 10, 130, 150, 170. 190, or 210 linked nucleotides. In some embodiments, the length of the intermediary sequence is about 30 to about 210, about 60 to about 210, about 90 to about 210, about 120 to about 210, about 150 to about 210, about 180 to about 210, about 30 to about 180, about 60 to about 180, about 90 to about 180, about 120 to about 180. or about 150 to about 180 linked nucleotides.

[0255] An intermediary’ sequence may' also comprise or form a secondary structure (e.g , one or more hairpin loops) that facilitates the binding of an effector protein to a guide nucleic acid and / or modification activity of an effector protein on a target nucleic acid (e.g., a hairpin region). An intermediary’ sequence may comprise from 5’ to 3’, a 5’ region, a hairpin region, and a 3’ region. In some embodiments, the 5’ region may hybridize to the 3’ region. In some embodiments, the 5’ region of the intermediary’ sequence does not hybridize to the 3‘ region.

[0256] In some embodiments, the hairpin region may comprise a first sequence, a second sequence that is reverse complementary to the first sequence, and a stem-loop linking the first sequence and the second sequence. In some embodiments, an intermediary sequence comprises a stem-loop structure comprising a stem region and a loop region. In some embodiments, thestem region is 4 to 8 linked nucleotides in length Tn some embodiments, the stem region is 5 to 6 linked nucleotides in length. In some embodiments, the stem region is 4 to 5 linked nucleotides in length. In some embodiments, an intermediary sequence comprises a pseudoknot (e.g., a secondary structure comprising a stem at least partially hybridized to a second stem or half-stem secondary structure). An effector protein may interact with an intermediary sequence comprising a single stem region or multiple stem regions, in some embodiments, the nucleotide sequences of the multiple stem regions are identical to one another. In some embodiments, the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others. In some embodiments, an intermediary sequence comprises 1 , 2, 3. 4, 5 or more stem regions.Handle sequence

[0257] In some embodiments, compositions, systems and methods described herein comprise the nucleic acid, wherein the nucleic acid comprises a handle sequence. In some embodiments, the handle sequence comprises an intermediary sequence. In some embodiments, the intermediary sequence is at the 3’-end of the handle sequence. In some embodiments, the intermediary sequence is at the 5’- end of the handle sequence. In some embodiments, the handle sequence further comprises one or more of linkers and repeat sequences. In some embodiments, the linker comprises a sequence of 5’-GAAA-3’ (SEQ ID NO: 9695). In some embodiments, the intermediary sequence is 5’ to the repeat sequence. In some embodiments, the intermediary' sequence is 5’ to the linker. In some embodiments, the intermediary sequence is 3’ to the repeat sequence. In some embodiments, the intermediary sequence is 3 ‘ to the linker. In some embodiments, the repeat sequence is 3’ to the linker. In some embodiments, the repeat sequence is 5’ to the linker.

[0258] In some embodiments, a sgRNA may include a handle sequence having a hairpin region, as well as a linker and a repeat sequence. The sgRNA having a handle sequence can have a hairpin region positioned 3’ of the linker and / or repeat sequence. The sgRNA having a handle sequence can have a hairpin region positioned 5’ of the linker and / or repeat sequence. The hairpin region may include a first sequence, a second sequence that is reverse complementary to the first sequence, and a stem-loop linking the first sequence and the second sequence.[02591 hi some embodiments, an effector protein may recognize a secondary structure of a handle sequence. In some embodiments, at least a portion of the handle sequence interacts with an effector protein described herein Accordingly, in some embodiments, at least a portion ofthe intermediary sequence interacts with the effector protein described herein. In some embodiments, both, at least a portion of the intermediary sequence and at least a portion of the repeat sequence, interacts with the effector protein. In general, the handle sequence is capable of interacting (e.g., non-covalent binding) with any one of the effector proteins described herein.

[0260] In some embodiments, the handle sequence of a sgRNA comprises a stem-loop structure comprising a stem region and a loop region. In some embodiments, the stem region is 4 to 8 linked nucleotides in length. In some embodiments, the stem region is 5 to 6 linked nucleotides in length. In some embodiments, the stem region is 4 to 5 linked nucleotides in length. In some embodiments, the sgRNA comprises a pseudoknot (e.g., a secondary' structure comprising a stem at least partially hybridized to a second stem or half-stem secondary structure). An effector protein may recognize a sgRNA comprising multiple stem regions. In some embodiments, the nucleotide sequences of the multiple stem regions are identical to one another. In some embodiments, the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others. In some embodiments, the sgRNA comprises at least 2, at least 3. at least 4, or at least 5 stem regions.

[0261] A handle sequence may include deoxyribonucleosides, ribonucleosides, chemically modified nucleosides, or any combination thereof. In some embodiments, a length of the handle sequence is at least 30, 50, 70, 90, 1 10, 130, 150, 170. 190, or 210 linked nucleotides. In some embodiments, a length of the handle sequence is not greater than 30, 50, 70, 90, 110, 130, 150, 170, 190. or 210 linked nucleotides. In some embodiments, the length of the handle sequence is about 30 to about 210. about 60 to about 210, about 90 to about 210. about 120 to about 210, about 150 to about 210, about 180 to about 210, about 30 to about 180, about 60 to about 180, about 90 to about 180, about 120 to about 180, or about 150 to about 180 linked nucleotides.

[0262] In some embodiments, the length of a handle sequence in a sgRNA is not greater than50, 56, 66, 67, 68, 69, 70, 71, 72, 73, 95, or 105 linked nucleotides. In some embodiments, the length of a handle sequence in a sgRNA is about 30 to about 120 linked nucleotides. In some embodiments, the length of a handle sequence in a sgRNA is about 50 to about 105, about 50 to about 95, about 50 to about 73, about 50 to about 71, about 50 to about 70, or about 50 to about 69 linked nucleotides. In some embodiments, the length of a handle sequence in a sgRNA is 56 to 105 linked nucleotides, from 56 to 105 linked nucleotides, 66 to 105 linked nucleotides, 67 to 105 linked nucleotides, 68 to 105 linked nucleotides, 69 to 105 linked nucleotides, 70 to 105 linked nucleotides, 71 to 105 linked nucleotides, 72 to 105 linked nucleotides, 73 to 105linked nucleotides, or 95 to 105 linked nucleotides. In some embodiments, the length of a handle sequence in a sgRNA is 40 to 70 nucleotides. In some embodiments, the length of a handle sequence in a sgRNA is 50, 56, 66, 67, 68. 69, 70, 71, 72, 73, 95, or 105 linked nucleotides.Single Nucleic Acid Systems

[0263] In some embodiments, compositions, systems, and methods described herein comprise a single nucleic acid system comprising a guide nucleic acid or a nucleotide sequence encoding the guide nucleic acid, and one or more effector proteins or a nucleotide sequence encoding the one or more effector proteins. In some embodiments, a first region (FR) of the guide nucleic acid non-covalently interacts with the one or more polypeptides described herein. In some embodiments, a second region (SR) of the grade nucleic acid hybridizes with a target sequence of the target nucleic acid. In the single nucleic acid system having a complex of the guide nucleic acid and the effector protein, the effector protein is not transactivated by the guide nucleic acid. In other words, activity’ of effector protein does not require binding to a second non-target nucleic acid molecule. An exemplary guide nucleic acid for a single nucleic acid system is a crRNA or a sgRNA. crUNA

[0264] In some embodiments, guide nucleic acid comprises a crRNA comprising a spacer sequence(s) and a repeat sequence(s) present within the same polynucleotide molecule. In some embodiments, the spacer sequence is adjacent to the repeat sequence. In some embodiments, the spacer sequence follows the repeat sequence in a 5’ to 3’ direction. In some embodiments, the spacer sequence precedes the repeat sequence in a 5’ to 3’ direction. In some embodiments, the spacer(s) and repeat sequence(s) are linked directly to one another. In some embodiments, a linker is present between the spacerfs) and repeat sequence(s). Linkers may be any suitable linker.

[0265] In some embodiments, a crRNA is useful as a single nucleic acid system for compositions, methods, and systems described herein or as part of a single nucleic acid system for compositions, methods, and systems described herein In some embodiments, a crRNA is useful as part of a single nucleic acid system for compositions, methods, and systems described herein. In such embodiments, a single nucleic acid system comprises a guide nucleic acid comprising a crRNA wherein, a repeat sequence of a crRNA is capable of connecting a crRN A to an effector protein. In some embodiments, a single nucleic acid system comprises a guidenucleic acid comprising a crRNA linked to another nucleotide sequence that is capable of being non-covalently bond by an effector protein. In such embodiments, a repeat sequence of a crRNA can be linked to an intermediary RNA. In some embodiments, a single nucleic acid system comprises a guide nucleic acid comprising a crRNA and an intermediary RNA.

[0266] In some embodiments, a crRNA is sufficient to form complex with an effector protein(e.g., to form an RNP) through the repeat sequence and direct the effector protein to a target nucleic acid sequence through the spacer sequence. In some embodiments, the repeat sequence in the crRNA polynucleotide hybridizes with a tracr sequence present in a separate polynucleotide. In some embodiments, the hybridization with the tracr sequences permits formation of an RNP complex with an effector protein

[0267] A crRNA may include deoxyribonucleosides, ribonucleosides, chemically modified nucleosides, or any combination thereof. In some embodiments, a crRNA comprises about: 10, 11. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25. 26. 27. 28. 29. 30. 31. 32. 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52. 53. 54. 55. 56. 57. 58. 59. or 60 linked nucleotides. In some embodiments, a crRNA comprises at least: 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60 linked nucleotides. In some embodiments, the length of the crRNA is about 20 to about 120 linked nucleotides. In some embodiments, the length of a crRNA is about 20 to about 100, about 30 to about 100, about 40 to about 100, about 40 to about 90, about 40 to about 80, about 40 to about 70, about 40 to about 60, about 40 to about 50, about 50 to about 90, about 50 to about 80, about 50 to about 70, or about 50 to about 60 linked nucleotides. In some embodiments, the length of a crRNA is about 20, about 25, about 30, about 35, about 40, about 45. about 50, about 55. about 60, about 65, about 70 or about 75 linked nucleotides. sgRNA

[0268] In some embodiments, a guide nucleic acid comprises a single guide RNA (sgRNA). In some embodiments, the guide nucleic acid is a sgRNA. The combination of a spacer sequence (e.g.. a nucleotide sequence that hybridizes to a target sequence in a target nucleic acid) with a handle sequence may be referred to herein as a single guide RNA (sgRNA), wherein the spacer sequence and the handle sequence are covalently linked. In some embodiments, the spacer sequence and handle sequence are linked by a phosphodiester bond. In some embodiments, the spacer sequence and handle sequence are linked by one or more linked nucleotides. In some embodiments, a guide nucleic acid may comprise a spacer sequence, a repeat sequence, or handle sequence, or a combination thereof. In some embodiments, the handle sequence may comprise a portion of, or all of, a repeat sequence. Ingeneral, a sgRNA comprises a first region (FR) and a second region (SR), wherein the FR comprises a handle sequence and the SR comprises a spacer sequence.

[0269] In some embodiments, the compositions comprising a guide RNA and an effector protein without a tracrRNA (e.g., a single nucleic acid system), wherein the guide RNA is a sgRNA. A sgRNA may include deoxyribonucleosides, ribonucleosides, chemically modified nucleosides, or any combination thereof. A sgRNA may also include a nucleotide sequence that forms a secondary structure (e.g., one or more hairpin loops) that facilitates the binding of an effector protein to the sgRNA and / or modification activity of an effector protein on a target nucleic acid (e.g.. a hairpin region). Such a sequence can be contained within a handle sequence as described herein.

[0270] In some embodiments, a sgRNA comprises one or more of one or more of a handle sequence, an intermediary' sequence, a crRNA, a repeat sequence, a spacer sequence, a linker, or combinations thereof. For example, a sgRNA comprises a handle sequence and a spacer sequence: an intermediary sequence and an crRNA; an intermediary sequence, a repeat sequence and a spacer sequence, and the like.

[0271] In some embodiments, a sgRNA comprises an intermediary sequence and an crRNA.In some embodiments, an intermediary sequence is 5' to a crRNA in an sgRNA. In some embodiments, a sgRNA comprises a linked intermediary sequence and crRNA. In some embodiments, an intermediary sequence and a crRNA are linked in an sgRNA directly (e g., covalently linked, such as through a phosphodiester bond) In some embodiments, an intermediary sequence and a crRNA are linked in an sgRNA by any suitable linker, examples of which are provided herein.

[0272] In some embodiments, a sgRNA comprises a handle sequence and a spacer sequence.In some embodiments, a handle sequence is 5’ to a spacer sequence in an sgRNA. In some embodiments, a sgRNA comprises a. linked handle sequence and spacer sequence. In some embodiments, a handle sequence and a spacer sequence are linked in an sgRNA directly (e.g., covalently linked, such as through a phosphodiester bond) In some embodiments, a handle sequence and a spacer sequence are finked in an sgRNA by any suitable linker, examples of which are provided herein.

[0273] In some embodiments, a sgRNA comprises an intermediary' sequence, a repeat sequence, and a spacer sequence. In some embodiments, an intermediary sequence is 5' to a repeat sequence in an sgRNA. In some embodiments, a sgRNA comprises a linked intermediary sequence and repeat sequence In some embodiments, an intermediary sequenceand a repeat sequence are linked in an sgRNA directly (e.g., covalently linked, such as through a phosphodiester bond) In some embodiments, an intermedian- sequence and a repeal sequence are linked in an sgRNA by any suitable linker, examples of which are provided herein. In some embodiments, a repeat sequence is 5' to a spacer sequence in an sgRNA. In some embodiments, a sgRNA comprises a linked repeat sequence and spacer sequence. In some embodiments, a repeat sequence and a spacer sequence are linked in an sgRNA directly (e.g., covalently linked, such as through a phosphodi ester bond) In some embodiments, a repeat sequence and a spacer sequence are linked in an sgRNA by any suitable linker, examples of which are provided herein.

[0274] An exemplary’ handle sequence in a sgRNA may comprise, from 5‘ to 3’, a 5‘ region, a hairpin region, and a 3’ region. In some embodiments, the 5’ region may hybridize to the 3’ region. In some embodiments, the 5’ region does not hybridize to the 3’ region. In some embodiments, the 3" region is covalently linked to a spacer sequence (e.g.. through a phosphodiester bond) In some embodiments, the 5’ region is covalently linked to a spacer sequence (e.g. , through a phosphodiester bond).Dual Nucleic Add Systems

[0275] In some embodiments, compositions, systems and methods described herein comprise a dual nucleic acid system comprising a crRNA or a nucleotide sequence encoding the crRNA, a tracrRNA or a nucleotide sequence encoding the tracrRNA, and one or more effector proteins or a nucleotide sequence encoding the one or more effector proteins, wherein the crRNA and the tracrRNA are separate, unlinked molecules, wherein a repeat hybridization region of the tracrRNA is capable of hybridizing with an equal length portion of the crRNA to form a tracrRNA-crRNA duplex, wherein the equal length portion of the crRNA does not include a spacer sequence of the crRNA, and wherein the spacer sequence is capable of hybridizing to a target sequence of the target nucleic acid. In the dual nucleic acid system having a complex of the guide nucleic acid, tracrRNA, and the effector protein, the effector protein is transactivated by the tracrRNA. In other words, in a dual nucleic acid system, acti vity of the effector protein requires binding to a tracrRNA molecule.

[0276] In some embodiments, a repeat hybridization sequence is at the 3’ end of a tracrRNA. In some embodiments, a repeat hybridization sequence may have a length of about 1 , about 2, about 3, about 4, about 5, about 6, about 7, about 8. about 9. about 10, about 12, about 14, about 16, about 18, or about 20 linked nucleotides. In some embodiments, the length of the repeat hybridization sequence is 1 to 20 linked nucleotides.

[0277] In some embodiments, systems, compositions, and methods comprise a crRNA or a use thereof. In general, a crRNA comprises a first region (FR) and a second region (SR), wherein the FR of the crRNA comprises a repeat sequence, and the SR of the crRNA comprises a spacer sequence. In some embodiments, the repeat sequence and the spacer sequences are directly connected to each other (e g.. covalent bond (phosphodi ester bond)). In some embodiments, the repeat sequence and the spacer sequence are connected by a linker.|0278] In some embodiments, systems, compositions, and methods comprise a tracrRNA or a use thereof. In some embodiments, systems, compositions, and methods do not comprise a tracrRNA or a use thereof, A tracrRNA and / or tracrRNA-crRN A duplex may form a secondary structure that facilitates the binding of an effector protein to a tracrRNA or a tracrRNA-crRN A.In some embodiments, the secondary structure modifies activity of the effector protein on a target nucleic acid. In some embodiments, the secondary structure comprises a stem-loop structure comprising a stem region and a loop region. In some embodiments, the stem region is 4 to 8 linked nucleotides in length In some embodiments, the stem region is 5 to 6 linked nucleotides in length. In some embodiments, the stem region is 4 to 5 linked nucleotides in length. In some embodiments, the secondary structure comprises a pseudoknot (e g., a secondary structure comprising a stem at least partially hybridized to a second stem or halfstem secondary structure). An effector protein may recognize a secondary7structure comprising multiple stem regions. In some embodiments, nucleotide sequences of the multiple stem regions are identical to one another. In some embodiments, the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others. In some embodiments, the secondary structure comprises at least two, at least three, at least four, or at least five stem regions. In some embodiments, the secondary' structure comprises one or more loops. In some embodiments, the secondary7structure comprises at least one, at least two, at least three, at least four, or at least five loops.Guide nucleic acids for precision editing

[0279] In some embodiments, the present disclosure provides guide nucleic acids for use in combination with the effector proteins, RDDPs, and fusion proteins thereof described herein for precision editing of a target nucleic acids sequence. In some embodiments, guide nucleic acids for use in precision editing comprise a spacer sequence, a repeat sequence, a primer binding sequence, and a template sequence. In some embodiments, guide nucleic acids for use in precision editing comprise one or more linkers between one or more components of the guide nucleic acids. In some embodiments, a spacer sequence, a repeat sequence, a primer bindingsequence, and a template sequence are comprised in a single polynucleotide, referred to herein as an extended guide RNA (rtgRNA). See e.g., FIG. 3 and FIG. 4. In some embodiments, a spacer sequence, a repeat sequence, a primer binding sequence, and a template sequence are comprised in two polynucleotides - the spacer and repeat sequence comprised in a first polynucleotide and the primer binding sequence and the template sequence comprised in a second polynucleotide, referred to herein as a split RNA. See e.g , FIG. 5A, FIG. 5B, and FIG. 6.Template RNA

[0280] In some embodiments, compositions, systems, and methods described herein comprise a template RNA (retRNA), wherein the template RNA (retRN A ) comprises a primer binding sequence and a template sequence. The template RNA (retRNA) may also be referred to as an extension of a guide RNA. The extension may comprise 1 , 2, 3. 4, 5, 6. 7, 8, 9. 10, 1 1 , 12, 13, 14. 15. 16, 17, 18, 19, or 20 nucleotides. The extension may comprise more than 10 nucleotides, in some embodiments the extension is 10-20 nucleotides. In some embodiments the extension is 20-30 nucleotides. In some embodiments the extension is 30-40 nucleotides. In some embodiments the extension is 40-50 nucleotides. In some embodiments the extension is 50-60 nucleotides. In some embodiments the extension is 70-80 nucleotides. In some embodiments the extension is 80-90 nucleotides. In some embodiments the extension is 90-100 nucleotides. In some embodiments the extension is 100-150 nucleotides. The extension may be processed during guide RNA formation.[0281 j In some embodiments, the retRNA, the spacer sequence, and the repeat sequence are comprised in the same polynucleotide (e.g, an rtgRNA). In some embodiments, the spacer sequence and repeat sequence are comprised in a first polynucleotide and the retRNA is comprised in a second polynucleotide (e g., a split RNA system).

[0282] In some embodiments, the primer binding sequence hybridizes to a primer sequence on the non-target strand of the target dsDNA molecule. In some embodiments, the primer binding sequence hybridizes to a primer sequence on the target strand of the target dsDNA molecule. In some embodiments, the spacer sequence is complementary7to the target sequence on the target strand of the dsDNA molecule, and the primer binding sequence and / or the template sequence is complementary to a primer sequence on the non-target strand of the target dsDNA molecule. In some embodiments, the spacer sequence is complementary' to the target sequence on the non-target strand of the dsDNA molecule, and the primer binding sequence and'' or thetemplate sequence is complementary to a primer sequence on the target strand of the target dsDNA molecule.

[0283] In some embodiments, the primer binding sequence is 4, 5, 6, 7, 8, 9, 10. 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides long. In some embodiments the template sequence is at least 10, 1 1 , 12. 13. 14, 15, 16, 17. 18, 19, 20, 21, 22, 23, 24, 25. 26. 27, 28, 29, 30. 31 , 32, 33,34 or 35 nucleotides long. In some embodiments, at least a portion of the PBS is complementary at least a portion of the target nucleic acid sequence that is 5' of the nucleotide at position 13 relative to the PAM sequence. Such embodiments are particularly useful when used in a system comprising a CasM.265466 or CasPhi effector protein.

[0284] The template sequence may comprise one or more nucleotides having a different nucleobase than that of a nucleotide at the corresponding position in the target nucleic acid when a spacer sequence of the guide RNA and the target sequence are aligned for maximum identity. The one or more nucleotides may be contiguous. The one or more nucleotides may not be contiguous. The one or more nucleotides may each independently be selected from guanine, adenine, cytosine and thymine.Extended guide RNA (rtgRNA) systems

[0285] In some embodiments, the present disclosure provides extended guide nucleic acids (rtgRNA) and an effector protein and RDDP, or fusion protein thereof described herein, or nucleic acids encoding the same, wherein the spacer sequence, repeat sequence, template sequence, and primer binding sequence are each comprised in a single polynucleotide.

[0286] In some embodiments, the rtgRNA comprises, from 5‘ to 3’, a template sequence, a primer binding sequence, a repeat sequence, and a spacer sequence, optionally wherein a linker sequence is located between the primer binding sequence and the repeat sequence. See e.g, FIG. 3. In some embodiments, the rtgRNA comprises, from 5’ to 3’, a repeat sequence, a spacer sequence, a template sequence, and a primer binding sequence, optionally wherein a linker sequence is located between the template sequence and the spacer sequence. See e.g., FIG. 4.Split gRNA systems

[0287] In some embodiments, the present disclosure provides a split gRNA system, comprising a first polynucleotide comprising a spacer sequence and a repeat sequence (e.g., a gRNA) and a second polynucleotide comprising a primer binding sequence and a template sequence (e.g., an retRNA), and an effector protein and RDDP. or fusion protein thereof described herein, or nucleic acids encoding the same.

[0288] In some embodiments, the first polynucleotide comprises a spacer sequence and a repeat sequence. In some embodiments, the first polynucleotide is a crRNA, as described above. In some embodiments, the first polynucleotide comprises a spacer sequence and a handle sequence (also referred to herein as a scaffold sequence). In some embodiments, the first polynucleotide is an sgRNA, as described above.

[0289] In some embodiments, the second polynucleotide comprises a primer binding sequence and a template sequence (e.g., an retRNA). In some embodiments, the second polynucleotide further comprises an aptamer that is recognized by a biological tether protein linked to an RDDP described herein. In some embodiments, the aptamer is an MS2 aptamer (See Said et al (November 2009). "In vivo expression and purification of aptamer-tagged small RNA regulators". Nucleic Acids Research. 37 (20): el 33; and Johansson et al (1997). "RNA recognition by the MS2 phage coat protein". Seminars in Virology. 8 (3): 176-185). In some embodiments, the second polynucleotide comprises, from 5" to 3". an aptamer sequence, a template sequence, and a primer binding sequence See FIG. 5B. In some embodiments, the second polynucleotide comprises, from 5’ to 3’, template sequence, a primer binding sequence, and an aptamer sequence. See FIG. 5A. In FIG. SA and FIG. SB, an MS2 aptamer is shown on the 3’ and 5' ends of the retRNA. respectively. The MS2 aptamer is an exemplary sequence and an RNA sequence with the appropriate secondary structure may also be used instead of the aptamer.

[0290] In some embodiments, the second polynucleotide is circularized. See FIG. 6.Target Nucleic Acids and Samples

[0291] Described herein are compositions, systems and methods for modifying a target nucleic acid, wherein the target nucleic acid is a gene, a portion thereof a transcript thereof. In some embodiments, the target nucleic acid is a double stranded nucleic acid. In some embodiments, the double stranded nucleic acid is DNA. In some embodiments, die target nucleic acid comprises a eukaryotic gene. In some embodiments, the target nucleic acid comprises a human gene. In some embodiments, the target nucleic acid comprises an intron of a gene. In some embodiments, the target nucleic acid comprises a sequence that does not encode a protein (also referred to as a non-coding region).

[0292] In some embodiments, the target nucleic acid comprises a safe harbor locus. A “safe harbor locus” refers to a region of the genome that can maintain transgene expression without detrimentally altering the function of host ceils. Safe harbor loci are known in the art and include, but are not limited to AAVSI (FPPIR12C) gene, an ALB gene, an ANGPTL3 gene, anAPOC3 gene, an ASGR2 gene, a CCR5 gene, a FIX(F9)‘ gene, a G6PC gene, a Gys2 gene, an HGD gene, &LP(A) gene, a PCSK9 gene, a SERPINA1 gene, a TF gene, a TTR gene, ROGI1, ROGI2, and an intron thereof (See e.g., Sadelain, M.„ Papapetrou, E.P., and Bushman, ED. (2012). Safe harbours for the integration of new DNA in the human genome. Nat. Rev. Cancer 12, 51-58; and Aznauryan et al., 2022, Cell Reports Methods 2, 100154)

[0293] Criteria for identifying safe harbor loci in the human genome are also known in the art. See FIG. 1A of Aznauryan et al., 2022, Cell Reports Methods 2, 100154.

[0294] In some embodiments, an LSR or system comprising the same described herein are used to insert a donor nucleic acid sequence encoding a wildtype or functional protein (or functional domain thereof) into a safe harbor locus. In some embodiments, the donor nucleic acid encodes a wildtype or functional version of any one of the genes listed in TABLE 10. In some embodiments, an LSR or system comprising the same described herein is used to disrupt a gam of function allele or an allele encoding a misfolded protein.[0295j In certain embodiments, the target nucleic acid is a double stranded nucleic acid comprising a target strand find a non-target strand, wherein the target strand comprises a target sequence. In some embodiments, where a target strand comprises a target sequence, at least a portion of the guide nucleic acid is complementary to the target sequence on the target strand. In some embodiments, where the target nucleic acid is a double stranded nucleic acid comprising a target strand and a non-target strand, and wherein the target strand comprises a target sequence, at least a portion of the guide nucleic acid is complementary to the target sequence on the target strand. In some embodiments, a target nucleic acid comprises a PAM as described herein that is located on the non-target strand. Such a PAM described herein, in some embodiments, is adjacent (e.g., within 1 , 2, 3, 4. 5, 10, 20, 25 nucleotides) to the 5’ or 3’ end of the target sequence on the non-target strand of the double stranded DNA molecule. In some embodiments, such a PAM described herein is directly adjacent to the 5’or 3’ end of a target sequence on the non-target strand of the double stranded DNA molecule.

[0296] In some embodiments, an effector protein described herein, or a multimeric complex thereof, recognizes a PAM on a target nucleic acid. In some embodiments, multiple effector proteins of the multimeric complex recognize a PAM on a target nucleic acid. In some embodiments, only one effector protein of the multimeric complex recognizes a PAM on a target nucleic acid. In some embodiments, the PAM is 3’ to the spacer region of the crRNA. In some embodiments, the PAM is directly or adjacent (e.g., within 1 , 2, 3, 4, 5, 10, 20, 25nucleotides) 3’ or 5’ to the spacer region of the crRNA. In some embodiments, the PAM comprises a PAM sequence set forth in TABLE 4.

[0297] An effector protein or fusion protein of the present disclosure may cleave or nick a target nucleic acid within or near a protospacer adjacent motif (PAM) sequence of the target nucleic acid. In some embodiments, cleavage occurs within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 nucleotides of a 5 ’ or 3 ’ terminus of a PAM sequence. A target nucleic acid may comprise a PAM sequence adjacent to a sequence that is complementary to a guide nucleic acid spacer region.

[0298] In some embodiments, the target nucleic acid is in a cell. In some embodiments, the cell is a single-cell eukaryotic organism; a plant cell; an algal cell; a fungal cell; an animal cell; a cell of an invertebrate animal; a cell of a vertebrate animal such as fish, amphibian, reptile, bird, and mammal; or a ceil of a mammal such as a human, a non-human primate, an ungulate, a feline, a bovine, an ovine, and caprine. In exemplary embodiments, the cell is a eukaryotic cell. In exemplars' embodiments, the cell is a mammalian cell, a human cell, or a plant cell. In some embodiments, the cell is a human ceil.Methods of Treating a Disorder

[0299] Compositions, systems and methods disclosed herein may be useful for treating a disease in a subject by inserting a donor nucleic acid (transgene) into the genome utilizing an LSR system described herein, wherein the insertion results in production of a functional protein Thus, compositions, systems and methods disclosed herein are especially useful for treating loss of function (LOF) diseases. Non-limiting examples of LOF diseases are spinal muscular atrophy (SMA), Pompe disease, Duchene muscular dystrophy. Fanconi -Bickel syndrome, Danon disease, von Gierke's disease, Cori's disease, Forbes' disease, McArdle's disease, Hers' disease, Tarin's disease, Fanconi-Bickel syndrome. Aldolase A deficiency, cystic fibrosis, hemophilia A, hemophilia B, Giteiman syndrome. Tay-Sachs disease, sickle ceil anemia, familial hypercholesterolemia, hemochromatosis, Huntington’s disease, nephrogenic diabetes insipidus, familial glucocorticoid deficiency, familial hypogonadism, hypogonadism, euthyroid hyperthyroidism, central hypothyroidism, chondrodysplasia, benign familial hypocalciuric hypercalcemia, neonatal severe primary hyperparathyroidism, retinal degeneration, vitreoretinopathy , Hirschsprung disease, fragil e X syndrome, Gaucher’s Disease, glucose 6-phosphate dehydrogenase deficiency, holoprosencephaly, phenylketonuria, Friedreich’s ataxia, alpha- 1 antitrypsin deficiency. Niemann Pick’s disease. Angelman syndrome, and polycystic kidney disease.

[0300] In some embodiments, the methods comprise administration of an LSR system, or nucleic acid encoding the same, or composition comprising the same to a subject in need thereof. In some embodiments, methods comprise administering a cell that has been modified by a system described herein to a subject. By way of non-limiting example, the disease may be a cancer, an ophthalmological disorder, a neurological disorder, a neurodegenerative disease, a blood disorder, or a metabolic disorder, or a combination thereof. The disease may be an inherited disorder, also referred to as a genetic disorder.Donor Nucleic Adds

[0301] In some embodiments, a donor nucleic acid comprises a nucleic acid that is incorporated into a target nucleic acid or target sequence. In reference to a viral vector, the term donor nucleic acid refers to a sequence of nucleotides that will be or has been introduced into a cell following transfection of the viral vector. The donor nucleic acid may be introduced into the cell by any mechanism of the transfecting viral vector, including, but not limited to. integration into the genome of the cell or introduction of an episomal plasmid or viral genome via an integration sequence and an LSR. As another example, when used in reference to the activity of an effector protein or LSR, the term donor nucleic acid refers to a sequence of nucleotides that will be. or has been, inserted at the site of cleavage by the effector protein (cleaving (hydrolysis of a phosphodiester bond) of a nucleic acid resulting in a nick or double strand break -nuclease activity) or recombination mediated by the LSR.

[0302] Donor nucleic acids of any suitable size may be integrated into a target nucleic acid or genome. In some embodiments, the donor polynucleotide integrated into a genome is less than 3, about 3, 3.5, 4, 4.5, 5. 5.5. 6, 6.5, 7, 7.5, 8, 8.5. 9, 9.5. 10, 10.5, 11. 11.5, 12. 12.5, 13, 13.5, 14, 14 5, 15, 16, 17, 1 8, 19, 20, 25, 30. 35, 40, 45, 50, 100, 150. 200, 250, 300, 350, 400, 450, 500 kilobases in length. Tn some embodiments, donor nucleic acids are more than 500 kilobases (kb) in length.

[0303] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the SMN protein, or a mutation in the SMNI gene, in a subject with spinal muscular atrophy (SAIA).

[0304] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the acid alpha-glucosidase protein, or a mutation in the GAA gene, in a subject with Pompe disease.

[0305] In some embodiments the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the dystrophin protein, or a mutation in the DMD gene, in a subject with Duchene muscular dystrophy.

[0306] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the GI..UT2 protein, or a mutation in the SLC2A2 gene, in a subject with Fanconi -Bickel syndrome.

[0307] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the LAMP2 protein, or a mutation in the Mac-3 gene, in a subject with Danon disease.

[0308] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the glucose-6-phosphatase protein, a mutation in the G6PC gene, a mutation in the glucose-6-phosphate transporter, a mutation in the SLC37A4 gene, a mutation in the SLC17A3 protein, or a mutation in the SLC17A3 gene in a subject with von Gierke's disease.

[0309] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the glycogen debranching enzyme, or a mutation in the zlG'L gene, in a subject with Cori's disease or Forbes' disease.

[0310] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the glycogen phosphorylase protein, or a mutation in the PYGM gene, in a subject with McArdle's disease.

[0311] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of the function due to a mutation in the glycogen phosphorylase protein, or a mutation in the PYGL gene, in a subject with Hers' disease.

[0312] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the phosphofructokinase protein, or a mutation in the PFKM gene, in a subject with Tarui's disease.

[0313] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the GLUT2 protein, or a mutation in the SLC2A2 gene, in a subject with Fanconi-Bickel syndrome. [1)314] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the fructose-bisphosphate aldolase protein, or a mutation in the ALDOA gene, in a subject with Aldolase A deficiency .

[0315] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the CFTR protein, or a mutation in the CFTR gene, in a subject with cystic fibrosis.

[0316] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the factor VIII protein, or a mutation in the F8 gene, in a subject with hemophilia A.

[0317] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the factor IX protein, or a mutation in the F9 gene, in a subject with hemophilia B.

[8318] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the sodium chloride cotransporter protein, a mutation in the SLC12A3 gene, a mutation in the chloride voltage-gated channel Kb protein, or a mutation in the CLCNKB gene in a subject with Gitelnian syndrome.

[0319] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the Hexosaminidase A protein, or a mutation in the HEXA gene, in a subject with Tay-Sachs disease.

[0320] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the hemoglobin subunit beta protein, or a mutation in the HBB gene, in a subject with sickle cell anemia.

[0321] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the LDL receptor protein, a mutation in the LDLR gene, a mutation in the apolipoprotein B protein, a mutation in the APOB gene. a. mutation in the proprotein convertase subtilisin / Kexin type 9 (PCSK9) protein, or a mutation in the PCSK9 gene, in a subject with familial hypercholesterolemia,

[0322] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the solute carrier family 40 member 1 (SLC40AI) protein, or a mutation in the SLC40A1 gene, in a subject with hemochromatosis.

[0323] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the Huntingtin protein, or a mutation in the HIT gene, in a subject with Huntington's disease.

[0324] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the Vasopressin receptor 2 (V2R), a mutation in the A KPAGgene, a mutation in the Aquaporin-2 protein, or a mutation in the A QP2 gene, in a subject with nephrogenic diabetes insipidus.

[0325] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the melanocortin 2 receptor protein, a mutation in the MC2R gene, a mutation in the melanocortin 2 receptor accessory protein, a mutation in the MRAP gene, a mutation in the nicotinamide nucleotide transhydrogenase protein, or a mutation in the NNT gene, in a subject with familial glucocorticoid deficiency.

[0326] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of functi on due to a mutation in the KiSS l-derived peptide receptor protein, or a mutation in the KISSI gene, in a subject with familial hypogonadism.

[0327] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the prokineticin 2 protein, or a mutation in the PROK2 gene, in a subject with hypogonadism.

[0328] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the thy rotropin receptor protein, or a mutation in the TSHR gene, in a subject with euthyroid hyperthyroidism

[0329] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the immunoglobulin superfamily, member 1 protein, or a mutation in the IGSF1 gene, in a subject with central hypothyroidism.

[0330] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the collagen ty pe II alpha 1 chain protein, a mutation in the COL2A / gene, a mutation in the fibroblast growth factor receptor 3 protein, a mutation in the FGFR3 gene, a mutation in the sulfate transporter protein, or a mutation in the SLC26A2 gene, in a subject with chondrodysplasia.

[0331] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the calcium-sensing receptor protein, or a mutation in the CASR gene, in a subject with benign familial hypocalciunc hypercalcemia or neonatal severe primary' hyperparathyroidism.

[0332] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the rhodopsin protein, or a mutation in the RHO gene, in a subject with retinal degeneration.

[0333] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the frizzled class receptor 4 protein, a mutation in the FZD4gene, a mutation in the LDL receptor related protein 5 protein, a mutation in the LRP5 gene, a mutation in the norrin cystine knot growth factor protein, or a mutation in the NDP gene, in a subject with vitreoretinopathy.

[0334] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the ret proto-oncogene, a mutation in the RET gene, a mutation in the endothelin receptor type B protein, a mutation in the EDNRB gene, a mutation in endothelin 3 protein, or a mutation in the EDN3 gene, in a subject with Hirschsprung disease.

[0335] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the fragile X mental retardation protein, or a mutation in the FMRI gene, in a subject with fragile x syndrome.

[0336] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the glucosylceramidase beta 1 protein, or a mutation in the GBA1 gene, in a subject with Gaucher's Disease.

[0337] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the Glucose-6-phosphate dehydrogenase protein, or a mutation in the G6PD gene, in a subject with glucose 6-phosphate dehydrogenase deficiency.

[0338] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the Glucose-6-phosphate dehydrogenase protein, or a mutation in the G6PD gene, in a subject with glucose 6-phosphate dehydrogenase deficiency.

[0339] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the SIX hoineobox 3 protein, or a mutation in the SIX3 gene, m a subject with holoprosencephaly.

[0340] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the phenylalanine hydroxylase protein, or a mutation in the PAH gene, in a subject with phenylketonuria.

[0341] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the frataxin protein, or a mutation in the FXN gene, in a subject wi th F ri edrei ch ’ s ataxi a.

[0342] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the serpin family A member 1 protein, or a mutation in the SERPINA1 gene, in a subject with alpha- 1 antitrypsin deficiency.

[0343] In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the sphingomyelin phosphodiesterase 1 protein, a mutation inthe SMPD! gene, a mutation in the NPC intracellular cholesterol transporter 1 protein, a mutation in ths NPC1 gene, a mutation in ths NPC intracellular cholesterol transporter 2 protein, or a mutation in the NPC2 gene, in a subject with Niemann Pick's disease.[0344 j In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the ubiquitin protein ligase E3A protein, or a mutation in theUBE3A gene, in a subject with Angelman syndrome.103451 In some embodiments, the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the poly cystin 1 protein, a mutation in the PKD1 gene, a mutation in the polycystin 2 protein, a mutation in the PKD2 gene, a mutation in the PKHD1 ciliary71PT domain containing fibrocystin / polyductin protein, or a mutation in the PKHDi gene, in a subject with polycystic kidney disease.Chemical M odifications

[0346] Polypeptides (<?.g.. effector proteins, LSRs. and / or RDDPs) and nucleic acids (e.g.. engineered guide nucleic acids) described herein can be further modified as described throughout and as further described herein. Examples are modifications of interest that do not alter primary' sequence, including chemical denvatization of polypeptides, e.g, acylation, acetylation, carboxylation, amidation, etc. Also included are modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g. by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes.Also embraced are sequences that have phosphorylated amino acid residues, e.g. phosphotyrosine, phosphoserine, or phosphothreonine.

[0347] Modifications disclosed herein can also include modif ication of described polypeptides and / or engineered guide nucleic acids through any suitable method, such as molecular biological techniques and / or synthetic chemistry, to improve their resistance to proteolytic degradation, to change the target sequence specificity, to optimize solubility properties, to alter protein activity (e.g., transcription modulatory activity', enzymatic activity, etc.) or to render them more suitable. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g. D-amino acids or non-naturally occurring synthetic amino acids. D-amino acids may be substituted for some or all of the amino acid residues.Modifications can also include modifications with non-naturally occurring unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like.

[0348] Modifications can further include the introduction of various groups to polypeptides and / or engineered guide nucleic acids described herein. For example, groups can be introduced during synthesis or during expression of a polypeptide (e.g, an effector protein, LSR, base editing enzyme, and / or RDDP). which allow for linking to other molecules or to a surface. Thus, e.g , cysteines can be used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like.

[0349] Modifications can further include modification of nucleic acids described herein (e.g., engineered guide nucleic acids) to provide the nucleic acid with a new or enhanced feature. such as improved stability. Such modifications of a nucleic acid include a base modification, a backbone modification, a sugar modification, or combinations thereof, of one or more nucleotides, nucleosides, or nucleobases in a nucleic acid.

[0350] In some embodiments, nucleic acids (e.g.. engineered guide nucleic acids) described herein comprise one or more modifications comprising: 2’O-methyl modified nucleotides. 2’ Fluoro modified nucleotides, locked nucleic acid (LNA) modified nucleotides; peptide nucleic acid (PNA) modified nucleotides; nucleotides with phosphorothioate linkages; a 5’ cap (e.g, a 7-methylguanylate cap (m7G)). phosphorothioates, chiral phosphoro thioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3‘-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoraniidates including 3'-amino phosphoramidate and aminoalkyl phosphoraniidates, phosphorodiamidates, thionophosphor amidates, thionoalkylphosphonates . thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5’ to 5’ or 2' to 2' linkage; phosphorothioate and / or heteroatom intern ucleoside linkages, such as -CH2-NH-O-CH2-, -CH2-N(CHa)-O-CH2- (known as a methylene (methylimino) or MMI backbone), -CH?- O-N(CH3)-CH2-, -CH2-N(CH3)- N(CH3)-CH2- and -O-NK’H3)-CH2-CH2- (wherein the native phosphodiester internucleotide linkage is represented as -O-P("O)(OH)-O-CH2-); morpholino linkages (formed in part from the sugar portion of a nucleoside); morpholino backbones; phosphorodiamidate or other non-phosphodiester intemucleoside linkages; siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneiramo and methylenehydrazinobackbones: sulfonate and sulfonamide backbones; amide backbones: other backbone modifications having mixed N, O, S and CH? component parts; and combinations thereof.Lectors and Multiplexed Expression Vectors[G3S1] In some embodiments, compositions and systems provided herein comprise a vector system encoding a polypeptide (e.g. , an LSR, effector protein, base editing enzy me, an RDDP, and / or a fusion protein thereof) and / or a guide nucleic acid described herein. In some embodiments, compositions and systems provided herein comprise a vector system encoding a guide nucleic acid (e.g. , crRNA) described herein. In some embodiments, compositions and systems provided herein comprise a multi-vector system encoding an LSR, effector protein, an RDDP, and / or a fusion protein thereof and a guide nucleic acid described herein, wherein the guide nucleic acid and the LSR, effector protein, the RDDP, and / or the fusion protein thereof are encoded by the same or different vectors. In some embodiments, the guide and the engineered effector protein are encoded by different vectors of the system. In some embodiments, a nucleic acid encoding a polypeptide (e.g , an LSR, an effector protein, base editing enzyme, an RDDP, and / or a fusion protein thereof) comprises an expression vector. In some embodiments, a nucleic acid encoding a polypeptide is a messenger RNA. In some embodiments, an expression vector comprises or encodes an engineered guide nucleic acid. In some embodiments, the expression vector encodes the crRNA

[0352] In some embodiments, a vector can comprise or encode one or more regulatory elements. Regulatory elements can refer to transcriptional and translational control sequences, such as promoters, enhancers, poly adenylation signals, terminators, protein degradation signals, and the like, that provide for and / or regulate transcription of a non-coding sequence or a coding sequence and / or regulate translation of an encoded polypeptide. In some embodiments, a vector can comprise or encode for one or more additional elements, such as, for example, replication origins, antibiotic resistance (or a nucleic acid encoding the same), a tag (or a nucleic acid encoding the same), selectable markers, and the like.

[0353] Vectors described herein can encode a promoter - a regulatory region on a nucleic acid, such as a DNA sequence, capable of initiating transcription of a downstream (3' direction) coding or non-coding sequence. As used herein, a promoter can be bound at its 3' terminus to a nucleic acid the expression or transcription of which is desired, and extends upstream (5' direction) to include bases or elements necessary to initiate transcription or induce expression, which could be measured at a detectable level. A promoter can comprise a nucleotide sequence, referred to herein as a “promoter sequence”. A promoter sequence can include a transcriptioninitiation site, and one or more protein binding domains responsible for the binding of' transcription machinery, such as RNA polymerase. When eukaryotic promoters are used, such promoters can contain “TATA” boxes and “CAT” boxes. Various promoters, including inducible promoters, may be used to drive expression, i.e., transcriptional activation, of the nucleic acid of interest Accordingly, in some embodiments, the nucleic acid of interest can be operably linked to a promoter.]G354] Promotors can be any suitable type of promoter envisioned for the compositions, systems, and methods described herein. Examples include constitutively active promoters (e.g. , CMV promoter), inducible promoters (e.g. , heat shock promoter, tetracycline-regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc,), spatially restricted and / or temporally restricted promoters (e.g., a tissue specific promoter, a cell type specific promoter, etc,), etc. Suitable promoters include, but are not limited to: SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6), an enhanced U6 promoter, and a human Hl promoter (Hl). By transcriptional activation, it is intended that transcription will be increased above basal levels in the target cell by 10 fold, by 100 fold, or by 1000 fold, or more. In addition, vectors used for providing a nucleic acid that, when transcribed, produces a guide nucleic acid and / or a nucleic acid that encodes an effector protein io a cell may include nucleic acid sequences that encode for selectable markers in the target cells, so as to identify cells that have taken up the guide nucleic acid and / or an effector protein

[0355] In general, vectors provided herein comprise at least one premotor or a combination of promoters driving expression or transcription of one or more genome editing tools described herein. In some embodiments, the viral vector comprises a nucleotide sequence of a promoter. In some embodiments, the viral vector comprises two promoters. In some embodiments, the viral vector comprises three promoters. In some embodiments, the length of the promoter is less than about 500, less than about 400, or less than about 300 linked nucleotides. In some embodiments, the length of the promoter is at least 100 linked nucleotides. Non-limiting examples of promoters include CMV, 7SK. EFla, RPBSA, hPGK, EFS. SV40, PGK1, Ubc, human beta actin promoter, TRE, UAS, Ac5, Polyhedrin. CaMKIIa, GALI , Hl, TEF1 , GDS, ADH1, CaMV35S, Ubi, U6, MNDU3, MSCV, MND and CAG.

[0356] In some embodiments, the promoter is an inducible promoter that only drives expression of its corresponding gene when a signal is present, e.g., a hormone, a small molecule, a peptide. Non-limiting examples of inducible promoters are the T7 RNA polymerase promoter, the T3 RNA polymerase promoter, the Isopropyl-beta-D- thiogalactopyranoside (IPTG)-regulated promoter, a lactose induced promoter, a heat shock promoter, a tetracycline-regulated promoter (tetracycline-inducible or tetracyclme- repressibie), a steroid regulated promoter, a metal-regulated promoter, and an estrogen receptor-regulated promoter. In some embodiments, the promoter is an activation-inducible promoter, such as a CD69 promoter, as described further in Kulemzin et al.. (2019). BMC Med Genomics, 12:44. In some embodiments, the promoter for expressing effector protein is a muscle-specific promoter. In some embodiments, the muscle-specific promoter comprises Ck8e, SPC5-12, or Desmin promoter sequence. In some embodiments, the promoter for expressing effector protein is a ubiquitous promoter. In some embodiments, the ubiquitous promoter comprises MND or CAG promoter sequence.

[0357] In some embodiments, an effector protein, base editing enzyme, LSR, RDDP, or fusion protein comprising the same (or a nucleic acid encoding same) and / or a guide nucleic acid (or a nucleic acid that, when transcribed, produces same) are co-administered with a donor nucleic acid. Coadininistration can be contact with a target nucleic acid, administered to a ceil, such as a host cell, or administered as method of nucleic acid detection, editing, an d / 'or treatment as described herein, in a single vehicle, such as a single expression vector. In certain embodiments, an effector protein, base editing enzyme LSR, RDDP, or fusion protein comprising the same (or a nucleic acid encoding same) and / or agui de nucleic acid (or a nucleic acid that, when transcribed, produces same) are not co-administered with donor nucleic acid in a single v ehicle. In certain embodiments, an effector protein (or a nucleic acid encoding same), a guide nucleic acid (or a nucleic acid that, when transcribed, produces same), and / or donor nucleic acid are administered in one or more or two or more vehicles, such as one or more, or tw’O or more expression vectors.Viral Vectors|0358] An expression vector can be a viral vector. In some embodiments, a viral vector comprises a nucleic acid to be delivered into a host cell via a recombinantly produced virus or viral particle. The nucleic acid may be single-stranded or double stranded, linear or circular, segmented or non-segmented. The nucleic acid may comprise DNA, RNA, or a combination thereof. In some embodiments, the expression vector is an adeno-associated viral vector. Thereare a variety of viral vectors that are associated with various types of viruses, including but not limited to retroviruses (e g., lentiviruses and y-retroviruses), adenoviruses, arenaviruses, alphaviruses, adeno-associated viruses (AAVs), baculoviruses, vaccinia viruses, herpes simplex viruses and poxviruses. A viral vector provided herein can be derived from or based on any such virus. Often the viral vectors provided herein are an adeno-associated viral vector(AAV vector). Generally, an AAV vector has two inverted terminal repeats (ITRs). According, in some embodiments, the viral vector provided herein comprises two inverted terminal repeats of AAV. The DNA sequence in between the ITRs of an AAV vector provided herein may be referred to herein as the sequence encoding the genome editing tools or a transgene. 'These genome editing tools can include, but are not limited to, an effector protein, effector protein modifications (c.g, nuclear localization signal (NLS), poly A tail), guide nucleic acid(s), respective promoter(s), and a donor nucleic acid, or combinations thereof. In some embodiments, a nuclear localization signal comprises an entity' (e.g., peptide) that facilitates localization of a nucleic acid, protein, or small molecule to the nucleus, when present in a cell that contains a nuclear compartment.[03591 In general, viral vectors provided herein comprise at least one promotor or a combination of promoters driving expression or transcription of one or more genome editing tools described herein. In some embodiments, the length of the promoter is less than about 500, less than about 400. or less than about 300 linked nucleotides In some embodiments, the length of the promoter is at least 100 linked nucleotides. Non-limiting examples of promoters includeCMV, EFla, RPBSA, hPGK, EFS, SV40, PGK1, Ubc, human beta actin promoter, CAG, TRE, UAS, Ac5, Polyhedrm, CaMKIIa, GAL L HI, TEF L GDS, ADH1. CaMV35S, Ubi. U6, MNDU3. and MSCV. In some embodiments, the promoter is an inducible promoter that only drives expression of its corresponding gene when a signal is present, e.g., a hormone, a small molecule, a peptide. Non-limiting examples of inducible promoters are the T7 RNA polymerase promoter, the T3 RNA polymerase promoter, the Isopropyl-beta-D- thiogalactopyranoside (IPTG)-regulated promoter, a lactose induced promoter, a heat shock promoter, a tetracycline-regulated promoter (tetracycline-inducible or tetracycline- repressible), a steroid regulated promoter, a metal-regulated promoter, and an estrogen receptor-regulated promoter In some embodiments, the promoter is an activation-inducible promoter, such as a CD69 promoter, as described further in Kulemzm el al. , (2019), BMC Med Genomics, 12:44.

[0360] In some embodiments, the coding region of the AAV vector forms an intramolecular double-stranded DNA template thereby generating an AAV vector that is a self-complementary AAV (scAAV) vector. In general, the sequence encoding the genome editing tools of an scAAV vector has a length of about 2 kb to about 3 kb. The scAAV vector can comprise nucleotide sequences encoding an effector protein, providing guide nucleic acids described herein, and a donor nucleic acid described herein. In some embodiments, the AAV vector provided herein is a self-inactivating AAV vector.

[0361] In some embodiments, an AAV vector provided herein comprises a modification, such as an insertion, deletion, chemical alteration, or synthetic modification, relative to a wild-type AAV vector

[0362] In some embodiments, the viral particle that delivers the viral vector described herein is an AAV. AAVs are characterized by their serotype. Non-limiting examples of AAV serotypes are AAV1. AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVS. AAV9, AAV10, AAV 11, AAV 12, scAAV, AAV-rhl O, chimeric or hybrid AAV, or any combination, derivative, or variant thereof.Producing AAV Particles[ 0363 [ The AAV particles described herein can be referred to as recombinant AAV (rAAV). Often, rAAV particles are generated by transfecting AAV producing cells with an AAV- containing plasmid earn ing the sequence encoding the genome editing tools, a plasmid that carries viral encoding regions, l.e., Rep and Cap gene regions; and a plasmid that provides the helper genes such as EIA, E1B, E2A, E4ORF6 and VA. In some embodiments, the AAV producing ceils are mammalian cells. In some embodiments, host cells for rAAV viral particle production are mammalian ceils. In some embodiments, a mammalian cell for rAAV viral particle production is a COS cell, a HEK293T cell, a HeLa cell, a KB cell, a derivative thereof, or a combination thereof. In some embodiments, rAAV virus particles can be produced in the mammalian cell culture system by providing the rAAV plasmid to the mammalian cell. In some embodiments, producing rAAV virus particles in a mammalian cell can comprise transfecting vectors that express the rep protein, the capsid protein, and the gene-of-interest expression construct flanked by the ITR sequence on the 5’ and 3' ends. Methods of such processes are provided in, for example, Naso el al., BioDrugs, 2017 Aug;31(4): 317-334 and Benskey el al., (2019), Methods Mol Biol.. 1937:3-26, each of which is incorporated by reference in their entireties.

[0364] In some embodiments, rAAV is produced in a non-mammalian cell. In some embodiments, rAAV is produced in an insect cell. In some embodiments, an insect cell for producing rAAV viral particles comprises a Sf9 cell. In some embodiments, production of rAAV virus particles in insect cells can comprise baculovirus. In some embodiments, production of rAAV virus particles in insect cells can comprise infecting the insect cells with three recombinant baculoviruses. one carrying the cap gene, one carrying the rep gene, and one carrying the gene-of-interest expression construct enclosed by an ITR on both the 5’ and 3’ end. In some embodiments, rAAV virus particles are produced by the One Bac system. In some embodiments, rAAV virus particles can be produced by the Two Bac system. In some embodiments, in the Two Bac system, the rep gene and the cap gene of the AAV is integrated into one baculovirus virus genome, and the ITR sequence and the gene-of-interest expression construct is integrated into another baculovirus virus genome. In some embodiments, in the One Bac system, an insect cell line that expresses both the rep protein and the capsid protein is established and infected with a baculovirus virus integrated with the ITR sequence and the gene-of-interest expression construct. Details of such processes are provided in, for example.Smith et. al,, (1983), Mol. Cell. Biol., 3(12):2156-65; Urabe et al., (2002), Hum. Gene, Then, 1;13(16): 1935-43; and Benskey etal., (2019), Methods Mol Biol., 1937:3-26, each of which is incorporated by reference in its entirely.Lipid partides

[0365] In some embodiments, compositions and systems provided herein comprise a lipid particle. In some embodiments, a lipid panicle is a lipid nanoparticle (LNP). In some embodiments, a lipid or a lipid nanoparticle can encapsulate an expression vector. In some embodiments, a lipid or a lipid nanoparticle can encapsulate the effector protein, the LSR, the RDDP, the sgRNA or crRNA, the nucleic acid encoding the effector protein, or LSR, or RDDPand / or the DNA molecule encoding the sgRNA or crRNA. LNPs are effective for delivery of nucleic acids. Beneficial properties of LNP include ease of manufacture, low cytotoxicity and immunogenicity, high efficiency of nucleic acid encapsulation and cell transfection, multi-dosing capabilities and flexibility of design (Kulkami et al , (2018) Nucleic Acid Therapeutics, 28(3): 146-157). In some embodiments, a method can comprise contacting a cell with an expression vector. In some embodiments, contacting can comprise electroporation, lipofection, or lipid nanoparticle (LNP) delivery of an expression vector.Methods and Formulations for Introducing Systems and Compositions into a Target Ceil

[0366] A guide nucleic acid (or a nucleic acid comprising a nucleotide sequence encoding same), an LSR, an RDDP, and / or an effector protein described herein can be introduced into a host cell by any of a variety of well-known methods As a non-limiting example, a guide nucleic acid, an LSR. an RDDP, and / or effector protein can be combined with a lipid. As another nonlimiting example, a guide nucleic acid, an LSR, an RDDP and / or effector protein can be combined with a particle or formulated into a particle.Methods of Editing

[0367] Methods of editing described herein may be employed to generate a genetically modified cell. The cell may be a eukaiyotic cell (e.g., a mammalian cell) or a prokaryotic ceil (e.g., an archaeal cell). The cell may be derived from a multicellular organism and cultured as a unicellular entity. The cell may comprise a heritable genetic modification, such that progeny cells derived therefrom comprise the heritable genetic mutation. The cell may be progeny of a genetically modified cell comprising a genetic modification of the genetically modified parent cell. A genetically modified cell may comprise a deletion, insertion, mutation, or non-native sequence relative to a wild-type version of the cell or the organism from which the cell was derived.

[6368] Methods may comprise contacting a cell or a subject with an effector protein, LSR, base editing enzyme, RDDP. fusion protein thereof, or any combination thereof. Methods may comprise contacting a cell with a nucleic acid encoding the effector protein, LSR, base editing enzyme, RDDP. fusion protein thereof, or combination thereof. Hie nucleic acid may be an expression vector. The nucleic acid may comprise a messenger RNA. Methods may comprise contacting a cell or a subject with an extended guide RNA or a portion thereof. Methods may comprise contacting a cell or a subject with a nucleic acid (e.g., DNA) encoding the extended guide RNA, or a portion thereof

[0369] In some embodiments, the present disclosure provides a mammalian cell comprising a system described herein, e.g, an RDDP described herein (or a fusion protein comprising the same), an effector protein described herein (or a fusion protein comprising the same), an LSR (or a fusion protein comprising the same), a base editing enzyme (or fusion protein comprising the same) and / or an rtgRNA. In some embodiments, the present disclosure provides a mammalian cell comprising one or more polynucleotides encoding one or more components of a system described herein, or one or more vectors comprising the same, e.g., a polynucleotideencoding an RDDP, base editing enzyme, or LSR described herein (or a fusion protein comprising the same) or vector comprising the same, a polynucleotide encoding an effector protein described herein (or a fusion protein comprising the same) or a vector comprising the same, and / or a polynucleotide encoding an rtgRNA or a vector comprising the same.

[0370] Methods of the disclosure may be performed in a subject. Compositions of the disclosure may be administered to a subject. A subject may be a human. A subject may be a mammal (e.g., rat, mouse, cow, dog, pig, sheep, horse). A subject may be a vertebrate or an invertebrate. A subject may be a laboratory animal. A subject may be a patient. A subject may be at risk of developing, suffering from, or displaying symptoms a disease or disorder as set forth in herein. The subject may have a mutation associated with a gene described herein The subject may display symptoms associated with a mutation of a gene described herein In some embodiments, a mutation comprises a point mutation or single nucleotide polymorphism (SNP), a chromosomal mutation, a copy number mutation, or any combination thereof. A point mutation optionally comprises a substitution, insertion, or deletion. In some embodiments, a mutation comprises a chromosomal mutation. A chromosomal mutation can comprise an inversion, a deletion, a duplication, or a translocation. In some embodiments, a mutation comprises a copy number variation. A copy number variation can comprise a gene amplification or an expanding trinucleotide repeat. In some embodiments, mutations may be as described herein.

[0371] Methods of the disclosure may be performed in a cell. A cell may be in vitro. A cell may be in vivo. A cell may be ex vivo. A cell may be an isolated cell. A cell may be a ceil inside of an organism. A ceil may be an organism. A cell may be a cell m a cell culture A cell may be one of a collection of cells. A cell may be a mammalian cell or derived from a mammalian cell. A cell may be a rodent cell or derived from a rodent ceil. A cell may be a human cell or derived from a human cell. A cell may be a eukatyotic cell or derived from a eukatyotic cell. A cell may be a pluripotent stem cell. A cell may be a plant cell or derived from a plant cell. A cell may be an animal cell or derived from an animal cell. A cell may be an invertebrate cell or derived from an invertebrate cell. A cell may be a vertebrate cell or derived from a vertebrate cell.

[0372] A cell may be from a specific organ or tissue. Non-limiting examples of organs and tissues from which a cell may be obtained or in which a cell may be located include: muscle, adipose, bone, adrenal gland, pituitary gland, thyroid gland, pancreas, testes, ovaries, uterus.heart, lung, aorta, smooth vasculature, endometrium, brain, neurons, spinal cord, kidney, liver, esophagus, stomach, intestine, colon, bladder, and spleen.

[0373] The tissue may be the subject's blood, bone marrow, or cord blood. The tissue may be heterologous donor blood, cord blood, or bone marrow. The tissue may be allogenic blood, cord blood, or bone marrow. In some embodiments, the cell is a stem cell Non-limiting examples of stem cells are hematopoietic stem cells, muscle stem cells (also referred to as myoblasts or muscle progenitor cells), and pluripotent stem cells (including induced pluripotent stem cells). In some embodiments, the cell is cell derived or differentiated from a pluripotent stem cell In some embodiments, the cell is a hepatocyte.

[0374] In some embodiments, the cell is an immune cell. Non-limiting examples of immune cells are lymphocytes (T cells, B cells, and NK cells), neutrophils, and monocytes / macrophages.Pharmaceutical Compositions and Modes of Administration

[0375] Disclosed herein, in some aspects, are pharmaceutical compositions for modifying a target nucleic acid in a cell or a subject, comprising any one of the effector proteins, LSRs, RDDPs, fusion proteins thereof, or guide nucleic acids as described herein and any combination thereof. Also disclosed herein, in some aspects, are pharmaceutical compositions comprising a nucleic acid encoding any one of the effector proteins, LSRs, RDDPs, fusion proteins thereof, or guide nucleic acids as described herein and any combination thereof. In some embodiments, pharmaceutical compositions comprise a plurality of guide nucleic acids Pharmaceutical compositions may be used to modify7a target nucleic acid or the expression thereof in a cell in vitro, in vivo or ex vivo.

[0376] In some embodiments, pharmaceutical compositions comprise one or more nucleic acids encoding an effector protein, LSR, base editing enzyme, RDDP, fusion protein thereof, a guide nucleic acid, or a combination thereof; and a pharmaceutically acceptable carrier or diluent. The effector protein, fusion effector protein, fusion partner protein, or combination thereof may be any one of those described herein. The one or more nucleic acids may comprise a plasmid. The one or more nucleic acids may comprise a viral vector. In some embodiments, the viral vector is a lenti viral vector. In some embodiments, the vector is an adeno-associated viral (AAV) vector. In some embodiments, compositions, including pharmaceutical compositions, comprise a viral vector encoding a effector proteins. LSRs. RDDPs. fusion proteins thereof and a guide nucleic acid, wherein at least a portion of the guide nucleic acid binds to the effector protein.

[0377] In some embodiments, pharmaceutical compositions comprise a virus comprising a viral vector encoding an effector protein, LSR RDDP, fusion protein thereof, a guide nucleic acid, or a combination thereof: and a pharmaceutically acceptable earner or diluent. The virus may be a lentivirus. The virus may be an adenovirus. The virus may be a non-replicating virus. The virus may be an adeno-associated virus (AAV). The viral vector may be a retroviral vector.Retroviral vectors may include gamma-retroviral vectors such as vectors derived from the Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV) or the Murine Stem cell Virus (MSCV) genome. Retroviral vectors may include lentiviral vectors such as those derived from the human immunodeficiency vims (HIV) genome. In some embodiments, the viral vector is a chimeric viral vector, comprising viral portions from two or more viruses In some embodiments, the viral vector is a recombinant viral vector.

[0378] In some embodiments, the viral vector is an AAV, The AAV may be any AAV known in the art. In some embodiments, the viral vector corresponds to a virus of a specific serotype. In some examples, the serotype is selected from an AAV1 serotype, an AAV2 serotype, AAV3 serotype, an AAV4 serotype, AAV5 serotype, an AAV6 serotype, AAV7 serotype, an AAV8 serotype, an AAV9 serotype, an AAV 10 serotype, an AAV 11 serotype, and an AAV12 serotype. In some embodiments the .AAV vector is a recombinant vector, a hybrid AAV vector, a chimeric AAV vector, a self-complementary AAV (scAAV) vector, a single-stranded AAV or any combination thereof. scAAV genomes are generally known in the art and contain both DNA strands which can anneal together to form double-stranded DNA.

[0379] In some embodiments, methods of producing delivery' vectors herein comprise packaging a nucleic acid, or transgene, encoding an effector protein and a nucleic acid that, when transcribed, produces a guide nucleic acid, or a combination thereof, into an AAV vector. In some embodiments, methods of producing the delivery vector comprises, (a) contacting a cell with at least one nucleic acid, or transgene that, when transcribed, produces a guide nucleic acid; at least one nucleic acid that encodes: (i) a Replication (Rep) gene; and (it) a Capsid (Cap) gene that encodes an AAV capsid protein; (b) expressing the AAV capsid protein in the cell; (c) assembling an AAV particle; and (d) packaging a Cas effector encoding nucleic acid into the AAV particle, thereby generating an AAV delivery vector. In some embodiments, promoters, stuffer sequences, and any combination thereof may be packaged in the AAV vector. In some embodiments, the AAV vector comprises a sequence encoding a guide nucleic acid. In some embodiments, the guide nucleic acid comprises a crRNA. In some embodiments, the guide nucleic acid is a crRNA In some embodiments, the guide nucleic acid comprises asgRNA. In some embodiments, the guide nucleic acid is a sgRNA. In some examples, the AAV vector can package 1, 2, 3, 4, or 5 nucleotide sequences encoding guide nucleic acids or copies thereof. In some examples, the AAV vector packages 1 or 2 nucleotide sequences encoding guide nucleic acids or copies thereof. In some embodiments, the AAV vector packages a nucleotide sequence encoding a first guide nucleic acid and a nucleotide sequence encoding a second guide nucleic acid, wherein the first guide nucleic acid and the second guide nucleic acid are the same. In some embodiments, the AAV vector packages a nucleotide sequence encoding a first guide nucleic acid and a nucleotide sequence encoding a second guide nucleic acid, wherein the first guide nucleic acid and the second guide nucleic acid are different. In some embodiments, the AAV vector comprises inverted terminal repeats, e g., a 5’ inverted terminal repeat and a 3’ inverted terminal repeat. In some embodiments, the inverted terminal repeat comprises inverted terminal repeats from AAV. In some embodiments, the inverted terminal repeat comprises inverted terminal repeats of ssAAV vector or scAAV vector. In some embodiments, the AAV vector comprises a mutated inverted terminal repeat that lacks a terminal resolution site.

[0380] In some embodiments, a hybrid AAV vector is produced by transcapsidation, e.g., packaging an inverted terminal repeat (IT R) from a first serotype into a capsid of a second serotype, wherein the first and second serotypes may be not the same. In some examples, the Rep gene and ITR from a first A AV serotype (e.g., AAV2) may be used in a capsid from a second AAV serotype (e.g , AAV 9), wherein the first and second AAV serotypes may be not the same. As anon-limiting example, a hybrid AAV serotype comprising the AAV2 ITRs and AAV9 capsid protein may be indicated AAV2 / 9. In some examples, the hybrid AAV delivery vector comprises an AAV2 / I , A AV 2 / 2, AAV 2 / 4, AAV2 / 5. A AV 2 / 8. or AAV2 / 9 vector.

[0381] In some embodiments, the AAV vector may be a chimeric AAV vector. In some embodiments, the chimeric AAV vector comprises an exogenous amino acid or an amino acid substitution, or capsid proteins from two or more serotypes In some examples, a chimeric AAV vector may be genetically engineered to increase transduction efficiency, selectivity, or a combination thereof.

[0382] In some examples, the delivery vector may be a eukaryotic vector, a prokaryotic vector (e.g. , a bacterial vector) a viral vector, or any combination thereof. In some embodiments, the delivery vehicle may be a non-viral vector. In some embodiments, the delivers' vehicle may be a plasmid. In some embodiments, the plasmid comprises DNA. In some embodiments, the plasmid comprises RNA. In some examples, the plasmid comprises circular double-strandedDNA. In some examples, the plasmid may be linear. In some examples, the plasmid comprises one or more genes of interest and one or more regulatory' elements In some examples, the plasmid comprises a bacterial backbone containing an origin of replication and an antibiotic resistance gene or other selectable marker for plasmid amplification in bacteria. In some examples, the plasmid may be a minicircle plasmid In some examples, the plasmid contains one or more genes that provide a selective marker to induce a target cell to retain the plasmid. In some examples, the plasmid may be formulated for delivery through injection by a needle carrying syringe. In some examples, the plasmid may be formulated for delivery via electroporation. In some examples, the plasmids may be engineered through synthetic or other suitable means known in the art. For example, in some embodiments, the genetic elements may be assembled by restriction digest of the desired genetic sequence from a donor plasmid or organism to produce ends of the DNA which may then be readily ligated to another genetic sequence.10383 | In some embodiments, the vector is a non-viral vector, and a physical method or a chemical method is employed for delivery into the somatic ceil. Exemplary physical methods include electroporation, gene gun. sonoporation, magnetofection, or hydrodynamic delivery. Exemplary' chemical methods include delivery’ of the recombinant polynucleotide via liposomes such as, cationic lipids or neutral lipids; dendrimers; nanoparti cies; or cellpenetrating peptides

[0384] In some embodiments, a fusion effector protein as described herein is inserted into a vector. In some embodiments, the vector comprises a transgene which comprises a nucleotide sequence of one or more promoters, enhancers, ribosome binding sues, RNA splice sites, polyadenylation sites, a replication origin, and / or transcriptional terminator sequences.

[0385] In some embodiments, the AAV vector comprises a self-processing array system for guide nucleic acid. Such a self-processing array system refers to a system for multiplexing, stringing together multiple guide nucleic acids under the control of a single promoter. In general, plasmids and vectors described herein comprise at least one promoter. In some embodiments, the promoters are constitutive promoters. In other embodiments, the promoters are inducible promoters. In additional embodiments, the promoters are prokary otic promoters (e.g, drive expression of a gene in a prokaryotic cell). In some embodiments, the promoters are eukary otic promoters, (e.g.. drive expression of a gene m a eukaryotic cell). Exemplary promoters include, but are not limited to, CMV, EFla, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, Ac5, polyhedron, CaMKIIa, GALI-10, TEF1, GDS, ADH1, CaMV35S,Ubi, Hl, U6, CaMV35S, SV40, CMV, 7SK, and HSV TK promoter. Tn some embodiments, the promoter is CMV, In some embodiments, the promoter is EFla. In some embodiments, the promoter is U6. In some embodiments, the promote is Hl. In some embodiments, the promoter is 7SK. In some embodiments, the promoter is ubiquitin. In some embodiments, vectors are bicistronic or polycistronic vector (e g., having or involving two or more loci responsible for generating a protein) having an internal ribosome entry site (IRES) is for translation initiation in a cap-independent manner.

[0386] In some embodiments, the AAV vector comprises a promoter for expressing effector proteins. LSRs, RDDPs. and / or fusion proteins thereof In some embodiments, the promoter for expressing effector proteins, LSRs, RDDPs, and / or fusion proteins thereof is a site-specific promoter. In some embodiments, the promoter for expressing effector proteins, LSRs, RDDPs, and / or fusion proteins thereof is a muscle-specific promoter. In some embodiments, the muscle-specific promoter comprises Ck8e, SPC5-12. or Desmin promoter sequence. In some embodiments, the promoter for expressing effector proteins, LSRs, RDDPs, and / or fusion proteins thereof is a ubiquitous promoter. In some embodiments, the ubiquitous promoter comprises MN D or CAG promoter sequence.

[0387] In some embodiments, the AAV vector comprises a staffer sequence. A staffer sequence can refer to a non-coding sequence of nucleotides that adjusts the length of the viral genome when inserted into a vector to increase packaging efficiency, increase overall viral titer during production, increase transfection efficacy, increase transfection efficiency, and / or decrease vector toxicity. In some embodiments, the stuffer sequence comprises 5' untranslated region. 3’ untranslated region or combination thereof. In some embodiments, a stuffer sequence serves no other functional purpose than to increase the length of the viral genome. In some embodiments, a stuffer sequence may increase the length of the viral genome as well as have other functional elements

[0388] In some embodiments, the 3' -untranslated region comprises a nucleotide sequence of an intron. In some embodiments, the 3 ’-untranslated region comprises one or more sequence elements, such as an intron sequence or an enhancer sequence. In some embodiments, the 3'- un translated region comprises an enhancer. In some embodiments, vectors comprise tin enhancer Enhancers are nucleotide sequences that have the effect of enhancing promoter activity. In some embodiments, enhancers augment transcription regardless of the orientation of their sequence. In some embodiments, enhancers activate transcription from a distance of several kilo basepairs. Furthermore, enhancers are located optionally upstream or downstreamof a gene region to be transcribed, and / or located within the gene, to activate the transcription. Exemplars' enhancers include, but are not limited to, WPRE; CMV enhancers; the R-LJ5' segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; the intron sequence between exons 2 and 3 of rabbit 0-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3). p 1527-31. 1981); and the genome region of human growth hormone (J Immunol..Vol. 155(3), p. 1286-95, 1995). In some embodiments, the enhancer is WPRE.|0389] In some embodiments, the AAV vector comprises one or more polyadenylation (poly A) signal sequences. In some embodiments, the polyadenlyation signal sequence comprises hGH poly A signal sequence. In some embodiments, the polyadenlyation signal sequence comprises sv40 poly A signal sequence.

[0390] Pharmaceutical compositions described herein may comprise a salt. Tn some embodiments, the salt is a sodium salt. In some embodiments, the salt is a potassium salt. In some embodiments, the salt is a magnesium salt. In some embodiments, the salt is NaCI. In some embodiments, the salt is KNO?. In some embodiments, the salt is Mgz+SO42”.

[0391] Non-limiting examples of pharmaceutically acceptable carriers and diluents suitable for the pharmaceutical compositions disclosed herein include buffers (e.g. , neutral buffered saline, phosphate buffered saline); carbohydrates (e.g., glucose, mannose, sucrose, dextran, mannitol); polypeptides or ammo acids (e.g, glycine); antioxidants; chelating agents (e.g, EDTA. glutathione); adjuvants (e.g. , aluminum hydroxide); surfactants (Polysorbate 80, Polysorbate 20, or Plutonic F68); glycerol; sorbitol; mannitol; polyethyleneglycol; and preservatives.

[0392] In some embodiments, pharmaceutical compositions are in the form of a solution (e.g., a liquid). The solution may be formulated for injection, e.g, intravenous or subcutaneous injection. In some embodiments, the pH of the solution is about 7, about 7.1 , about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, about. 8.5, about 8.6, about 8.7. about 8.8, about 8.9, or about 9. In some embodiments, the pH is 7 to 7.5, 7,5 to 8, 8 to 8.5, 8.5 to 9, or 7 to 8.5. In some embodiments, the pH of the solution is less than 7. In some embodiments, the pH is greater than 7.

[0393] In some embodiments, pharmaceutical compositions comprise an: effector protein, fusion effector protein, fusion partner, LSR, RDDP, base editing enzyme, a guide nucleic acid, or a combination thereof; and a pharmaceutically acceptable carrier or diluent. In some embodiments, pharmaceutical compositions comprise one or more nucleic acids encoding an: effector protein, fusion effector protein, fusion partner, LSR, RDDP, base editing enzyme, aguide nucleic acid, or a combination thereof; and a pharmaceutically acceptable carrier or diluent.Exemplary Systems

[0394] In some embodiments, the present disclosure provides a system for the modification of a target nucleic acid comprising (1 ) a recombinase or a polynucleotide encoding the same and a donor DNA vector comprising a first recombinase recognition sequence (RRS-1) and wherein the target nucleic acid comprises a second RRS (RRS-2) sequence.In some embodiments, the recombinase comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the sequences selected from SEQ ID NOs: 1-3918 and 9761-9763, the RRS-1 sequence comprises a nucleic acid sequence that is at least 90%, al least, 91 %, at least 92%, at least 93%. at least 94%, at least 95%, at least 96%, at least 97%. at least 98%. or at least 99% identical to any one of the sequences selected from SEQ ID NOs: 6442-8964, 9740-9760, and 9764-9791. and the RRS-2 sequence comprises a nucleic acid sequence that is at least 90%, at least, 91%, at least 92%, at least 93%. at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the sequences selected from SEQ ID NOs: 3919-6441, 9719-9739, and 9792-9819, wherein the recombinase, RRS-2. and RRS-2 are present in the same row in TABLE 1 or TABLE 2 In some embodiments, the recombinase comprises any one of SEQ ID NOs: 1-3918 and 9761- 9763, the RRS-1 comprises any one of SEQ ID NOs: 6442-8964, 9740-9760, and 9764-9791, and the RRS-2 comprises any one of SEQ ID NOs: 3919-6441, 9719-9739. and 9792-9819, wherein the recombinase. RRS-2 and RRS-2 are present in the same row in TABLE 1 or TABLE 2. In some embodiments, the recombinase consists of any one of SEQ ID NOs: 1- 3918 and 9761-9763, the RRS-1 consists of any one of SEQ ID NOs: 6442-8964, 9740-9760, and 9764-9791, and the RRS-2 consists of any one of SEQ ID NOs: 3919-6441, 9719-9739, and 9792-9819, wherein the recombinase, RRS-2 and RRS-2 are present in the same row in TABLE I or TABLE 2.

[0395] In some embodiments, the system further comprises an effector protein.

[0396] In some embodiments, the effector protein sequence comprises an ammo acid sequence that is at least 90%. at least, 91%, at least 92%, at least 93%, at least 94%. at least 95%, at least 96%, at least 97%, at least 98%. or at least 99% identical to any one of SEQ ID NOs: 9267- 9401 and 9698-9701. In some embodiments, the effector protein sequence comprises any oneof SEQ ID NOs: 9267-9401 and 9698-9701. In some embodiments, the effector protein sequence consists of any one of SEQ ID NOs: 9267-9401 and 9698-9701.

[0397] In some embodiments, the system further comprises an effector protein sequence and an RDDP sequence, or a fusion protein comprising an effector protein and an RDDP.

[0398] In some embodiments, the RDDP sequence comprises an amino acid sequence that is at least 90%, at least, 91%, at least 92%. at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 9402-9482, and the effector protein sequence comprises an amino acid sequence that is at least 90%, at least, 9! %. at least 92%. at least 93%. at least 94%. at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% identical to any one of SEQ ID NOs: 9267-9401 and 9698-9701. In some embodiments, the RDDP sequence comprises any one of SEQ ID NOs: 9402-9482, and the effector protein sequence comprises any one of SEQ ID NOs: 9267-9401 and 9698-9701. In some embodiments, the RDDP amino acid sequence consists of any one of SEQ ID NOs: 9402-9482, and the effector protein amino acid sequence consists of any one of SEQ ID NOs: 9267-9401 and 9698-9701.

[0399] In some embodiments, the RDDP-effector fusion comprises an amino acid sequence that is at least 90%, at least, 91%, at least 92%, at least 93%, at least 94%. at least 95%, at least 96%. at least 97%, at least 98%, or at least 99% identical to any one of the sequences selected from SEQ ID NOs: 9702-9713. In some embodiments, the RDDP-effector fusion protein comprises an amino acid sequence selected from SEQ ID NOs: 9702-9713. In some embodiments, RDDP-effector fusion consists of any one of the sequences selected from SEQ ID NOs: 9702-9713.

[0400] In some embodiments, the recombinase comprises an amino acid sequence that is at least 90%, at least, 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the sequences selected from SEQ ID NOs: 3, 16, 18, 20, 350, 441 , 454, 455. 569, 591. 620, 1105. 1 121. 1319, 1452, 1518, 1944, 1966, 2058, 2068. 2172, and 9761-9763. the RRS-I sequence comprises a nucleic acid sequence that is at least 90%, at least, 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the sequences selected from SEQ ID NOs: 9740-9791, and the RRS-2 sequence comprises a nucleic acid sequence that is at least 90%, at least, 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the sequences selected from SEQ ID NOs: 9719-9739 and 9793-9819, wherein therecombinase, the RRS-1 and RRS-2 sequences are on the same line in any of TABLES 13-15. In some embodiments, the recombinase comprises any one of the sequences selected from SEQ ID NOs: 3, 16, 18, 20, 350, 441, 454, 455, 569, 591. 620, 1105, 1121, 1319, 1452, 1518, 1944, 1966, 2058, 2068, 2172, and 9761-9763, the RRS-1 sequence comprises any one of the sequences selected from SEQ ID NOs: 9740-9791 , and the RRS-2 sequence comprises any one of the sequences selected from SEQ ID NOs: 9719-9739 and 9793-9819, wherein the recombinase, the RRS-1 and RRS-2 sequences are on the same line in any of TABLES 13-15. In some embodiments, the recombinase consists of any one of the sequences selected from SEQ ID NOs: 3, 16, 18, 20, 350, 441. 454, 455. 569, 591, 620, 1 105. 1121, 1319, 1452, 1518, 1944. 1966, 2058, 2068, 2172, and 9761-9763. the RRS-1 sequence consists of any one of the sequences selected from SEQ ID NOs: 9740-9791 , and the RRS-2 sequence consists of any one of the sequences selected from SEQ ID NOs: 9719-9739 and 9793-9819. wherein the recombinase, the RRS-1 and RRS-2 sequences are on the same line in any of TABLES 13-15.SEQUENCES AND TABLES |0401] TABLE 1 provides illustrative amino acid sequences of effector proteins, guide sequences, and PAM sequences that are useful in the compositions, systems and methods described herein. With regards to the PAM sequences: N is any’ nucleotide. V is A. C, or G: B is C, G, or T; R is A or G; Y is C or T; and S is G or C.

[0402] TABLEs 1 and 2 provide LSRs and related RRS-1 and RRS-2 sites. In some embodiments, more than one RRS sequence may be provided for a given I., SR In such embodiments, the provided RRS sequences refer to RRS sequences of varying length, each of which comprises a core RRS sequence. For example, the RRS-2 sequence for LSR 2955954 can be SEQ ID NO: 3934, 9719, or 9792. wherein each of SEQ ID NOs: 3934 and 9792, comprise the core sequence of SEQ ID NO: 9719.TABLE 1: LSRs, RRS-l / RRS-2 sites, and safe harbor target sequencesTABLE 2: LSRs, RRS-J / RRS-2 sitesTABLE 4. Exemplary Effector Protein Amino Acid Sequences, Exemplary Guide RepeatSequences, and Exemplary PAM Sequences

[0403] TABLE 5 provides exemplary ammo acid alterations relative to SEQ ID NO: 9401 useful in compositions, systems, and methods described herein.TABLE 5: Exemplary Amino Acid Alterations Relative to SEQ II) NO: 9-101

[0404] TABLE 6 provides exemplary' amino acid alterations relative to SEQ ID NO: 9278 useful in compositions, systems, and methods described herein.TABLE 6: Exemplary Amino Acid Alterations Relative to SEf? ID NO: 9278

[0405] TABLE 7 provides exemplary effector protein CasPhi.12 engineered variant sequences.TABLE 7: Exemplary Effector Protein CasPhi.12 Engineered Variant Amino AcidSequences|0406] TABLE 8 provides exemplary RDDP sequences. TABLE 9 provides exemplary engineered RDDP sequences.TABLE 8: Exemplary RDDP SequencesTABLE 9: Exemplary Engineered RDDPs

[0407] TABLE 10 provides exemplary target genes.| | I | | | | | | | | | | | I | | | | | | | | | | | I | | | I | | | I I | | | I | | | I | | | I ||[0408 j TABLE 11 provides exemplary' RDDP-Effector Protein Fusion Sequences.TABLE 11. Exemplary RDDP-Effector Protein Fusion SequencesEXAMPLES

[0409] The following examples are included for illustrative purposes only and are not intended to limit the scope of the disclosure.Example 1. LSR Identification

[0410] LSRs were identified from metagenomic data based on matches to the Recombinase and Resolvase Pfam domain HMMs. Additional LSRs were identified that matched the Recombinase Pfam and at least one other Pfam of interest. Open source software GeNomad was used to identify phage boundaries on the metagenomic contigs containing these LSRs. Shared palindromic sequences (from phage and genome itself) that occur around the phage boundaries and LSR boundaries were used to construct predicted attachment sites for eachLSR. These attachment sites are iteratively shortened (in silico) on both ends to predict the “minimal attachment site” with the highest palindromi cits'.Example 2. LSR Activity in Mammalian Ceils[04111 Assay overview: An activity assay to determine novel LSR functionality was performed in HEK293T cells transfected with 2 plasmids. LSRs tested includ...

Claims

CLAIMS1) A system for the modification of a target nucleic acid comprising: a) a recombinase or a nucleic acid encoding a recombinase; and b) a donor DNA vector comprising a first recombinase recognition sequence (R.R.S-1), wherein the recombinase comprises an ammo acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 1-3918 and 9761 -9763, and wherein the target nucleic acid comprises a second RRS (RRS-2).2) The system of claim I, wherein a) the recombinase comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NOs: 1-2523 and 9761-9763. b) the RRS-1 comprises a nucleotide sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs:6442-8964, 9740-9760, and 9764-9791, and c) the RRS-2 comprises a nucleotide sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 3919-6441, 9719-9739, and 9792-9819, wherein the recombinase, RRS-1 and RRS-2 are in the same row of TABLE 1 or TABLE 2.3) The system of claim 2, wherein the RRS-1 and / or RRS-2 is located in or near a safe harbor locus.4) The system of any of claims 2-3, wherein the safe harbor locus is near an AAVS1 (PPPIR12C) gene, an ALff gene, anANGPTL3 gene. anAPOCJ gene. anASGR2 gene, a CCR5 gene, a FIX (F9) gene, a G6PC gene, a GYS2 gene, an HGD gene, an LP(A) gene, a PCSK9 gene, a SERPINA / gene, a TF gene, and a TTR gene, or an intron thereof5) The system of any of claims 2-4, wherein the RRS-1 and / or RRS-2 is located in or near a target sequence of the human albumin gene (ALB) or AAVS1 gene.6) 'The system of any of claims 2-5, wherein the target sequence is selected from a nucleotide sequence that is at least 80%, at least 90%, at least 95% or 100% identical to asequence selected from SEQ ID NOs: 8965-9266, and wherein the target sequence is in the same row of TABLE 1 as the recombinase, RRS-1, and RRS-2.7) The system of any preceding claim, wherein the donor DNA vector is a circularized DMA molecule.8) Tire system of any preceding claim, wherein the recombinase or nucleic acid encoding the recombinase, and the donor DNA vector are combined in a single composition.9) ’The system of any preceding claim, wherein the nucleic acid encoding the recombinase comprises the donor DNA.10) A method of modifying a target nucleic acid comprising contacting the target nucleic acid with the components of the system of any one of claims 1-9.11) A method of modifying a target nucleic acid in a ceil, the method comprising contacting the cell with the components of the system of any one of claims 1-9.12) A composition for the modification of a target nucleic acid, the composition comprising a fusion protein or a nucleic acid encoding the fusion protein, wherein the fusion protein comprises: a) a recombinase comprises an ammo acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 1-3918 and 9761-9763; b) an effector protein; and c) an RNA dependent DNA polymerase (RDDP)13) The composition of claim 12, wherein the effector protein is a CRISPR associated (Cas) protein.14) The composition of claim 13, wherein the Cas protein is a Ty pe V Cas protein.15) The composition of claim 13, wherein the Cas protein comprises an amino acid sequence that is at least 75%, at least 80%. at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 9267-9401 and 9698-9701 .16) The composition of any one of claims 12-15, wherein the length of the Cas protein is350 to 550 ammo acids.17) The composition of any one of claims 12-15, wherein the length of the Cas protein is550 to 800 amino acids.18) The composition of any one of claims 12-17 , wherein the Cas protein comprises nickase activity.19) Tire composition of any one of claims 12-18, wherein the RDDP comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 9402-9482.20) The composition of any one of claims 12-19, comprising an extended guide nucleic acid, w -herein the extended guide nucleic acid comprises: i) a protein binding sequence, wherein the effector protein is capable of binding the protein binding sequence, ii) a spacer sequence that hybridizes to a first target sequence on a first strand of the target nucleic acid, iii) a primer binding sequence, optionally wherein a portion of the primer binding sequence hybridizes to a first portion of the second strand of the target nucleic acid: and iv) a template sequence that hybridizes to a second portion of the second strand of the target nucleic acid.21) The composition of any one of claims 12-19, comprising a first guide nucleic acid, wherein the extended guide nucleic acid comprises: i) a protein binding sequence, wherein the effector protein is capable of binding the protein binding sequence, ii) a spacer sequence that hybridizes to a first target sequence on a first strand of the target nucleic acid, iii) a primer binding sequence, optionally wherein a portion of the primer binding sequence hybridizes to a first portion of the first strand of the target nucleic acid; and iv) a template sequence that hybridizes to a second portion of the first strand of the target nucleic acid.22) Tire composition of claim 20 or 21. comprising a donor DNA vector, w herein the donor DNA vector comprises a first RRS (RRS-1).23) The composition of claim 20 or 21 , wherein the template sequence of the extended guide nucleic acid comprises at least a portion of a first or second RRS, and the target sequence optionally comprises a portion of the first or second RRS.24) The composition of any one of claims 12-22, comprising a second guide nucleic acid, wherein the guide nucleic acid comprises a protein binding sequence and a spacer sequence that hybridizes to a second target sequence on the second strand of the target nucleic acid.25) The composition of any one of claims 12-2.1, wherein the fusion protein comprises a nuclear localization signal.26) A method of modifying a target nucleic acid comprising contacting the target nucleic acid with the composition of any one of claims 12-25.27) A method of modifying a target nucleic acid in a ceil, the method comprising contacting the cell with the composition of any one of claims 12-25.28) A system for the modification of a target nucleic acid comprising: a) a recombinase or a nucleic acid encoding a recombinase; b) a donor DNA vector comprising a first recombinase recognition sequence (RRS-1), wherein the target nucl eic acid comprises a second RRS (RRS-2); and wherein i) the recombinase comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%. at least 99%, or 100% identical to SEQ ID NO: 3; the RRS-1 comprises or consists of an ammo acid sequence that is at least 70%, at least 75%. at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to any one of SEQ ID NOs: 6444 or 9766; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 3921 or 9794; fi) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 16; the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 6457, 9740. or 9764; and the RRS-2 comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 3934, 9719. or 9792; iii) the recombinase comprises or consists o f an ammo acid sequence that is at least 70%, at least 75%, at least 80%. at least 85%. at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 18; the RRS-1 comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%. at least 85%, at least 90%. at least 95%. at least 98%. at least 99%, or 100% identical to any one of SEQ ID NOs: 6459, 9743. or 9767; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%. at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 3936, 9722, or 9795; iv) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%. at least 99%. or 100% identical to SEQ ID NO: 20; the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%. at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 6461, 9741, or 9765; and the RRS-2 comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%. at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 3938, 9720, or 9793; v) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%. at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 350, the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 6791. 9756, or 9780; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%. at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 4268, 9735, or 9808: vi) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%. at least 99%, or 100% identical to SEQ ID NO: 441: the RRS-I comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%. at least 85%. at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 6882, 9757, 9781, or 9784 ; and the RRS-2 comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%. at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 4359. 9736, 9809, or 9812; vii) the recombinase comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 454; the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 6895, 9751 , or 9775; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%. at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 4372. 9730. or 9803; viii) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 455; the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 6896, 9750, or 9774; and the RRS-2 comprises or consistsof an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 4373, 9729, or 9802; ix) the recombinase comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%. at least 95%, at least 98%. at least 99%, or 100% identical to SEQ ID NO: 569; the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%. at least 85%, at least 90%, at least 95%, at least 98%. at least 99%, or 100% identical to any one of SEQ ID NOs: 7010, 9748, or 9772; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%. at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 4487. 9727, or 9800; x) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%. at least 99%, or 100% identical to SEQ ID NO: 591 : the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%. at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 7032, 9755, or 9779; and the RRS-2 comprises or consists of an ammo acid sequence that is at least 70%, at least 75 %, at least 80%, at least 85%, at least 90%. at least 95%. at least 98%. at least 99%, or 100% identical to any one of SEQ ID NOs: 4509, 9734, or 9807; xi) the recombinase comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%. at least 99%. or 100% identical to SEQ ID NO: 620; the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 7061. 9758, or 9782; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%. at least 99%, or 100% identical to any one of SEQ ID NOs: 4538, 9737, or 9810;xii) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%. at least 98%. at least 99%, or 100% identical to SEQ ID NO: 955; the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%. at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 7396 or 9791 ; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%. at least 95%. at least 98%. at least 99%, or 100% identical to any one of SEQ ID NOs: 4873 or 9819; xiii) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%. at least 99%. or 100% identical to SEQ ID NO: 1105; the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 7546, 9760. or 9783; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%. at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5023, 9739, or 9811; xiv) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%. at least 80%. at least 85%, at least 90%, at least 95%. at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1112; the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 7553. 9754, or 9778; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5030, 9733. or 9806; xv) the recombinase comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%. at least 99%, or 100% identical to SEQ ID NO:1162; the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs; 7603 or 9789; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%. at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5080 or 9817; xvi) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%. at least 80%. at least 85%. at least 90%, at least 95%. at least 98%, at least 99%. or 100% identical to SEQ ID NO: 1319; the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 7760, 9745, or 9769; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5237, 9724, or 9797; x vii) the recombinase comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%. at least 99%, or 100% identical to SEQ ID NO: 1452; the RRS-I comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 7893, 9749, or 9773; and the RRS-2. comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%. at least 98%. at least 99%, or 100% identical to any one of SEQ ID NOs: 5370. 9728. or 9801; xviii) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%. at least 99%. or 100% identical to SEQ ID NO: 1518; the RRS-I comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one ofSEQ ID NOs; 7959, 9752. or 9776; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%. at least 75%, at least 80%, at least 85%, at least 90%, at least 95%. at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5436, 9731, or 9804; xix) the recombinase coraprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%. at least 99%, or 100% identical to SEQ ID NO: 1735; the RRS-1 comprises or consists of an amino acid sequence that is at least 70%. at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 8176 or 9788; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%. at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5653 or 9816; xx) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%. at least 99%. or 100% identical to SEQ ID NO: 1944; the RRS-1 comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 8385. 9746, or 9770; and the RRS-2 comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%. at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5862, 9725, or 9798; xxi) the recombinase comprises or consists of an amino acid sequence that is at least 70%, al least 75%, at least 80%. at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1966; the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, al least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 8407, 9753, or 9777; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5884, 9732. or 9805; xxii) the recombinase comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%. at least 85%, at least 90%, at least 95%, at least 98%. at least 99%, or 100% identical to SEQ ID NO: 2050; the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 8491. 9744, or 9768; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%. at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5968, 9723. or 9796; xxiii) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%. at least 95%. at least 98%, at least 99%. or 100% identical to SEQ ID NO: 2068; the RRS-1 comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 8509, 9759. or 9790; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%. at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5986. 9738. or 9818; xxiv) the recombinase comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%. at least 85%, at least 90%, at least 95%, at least 98%. at least 99%, or 100% identical to SEQ ID NO: 2172; the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%. at least 85%. at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 8613, 9747, or 9771 ; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%. at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 6090, 9726, or 9799;xxv ) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%. at least 98%. at least 99%, or 100% identical to SEQ ID NO: 9761; the RRS-1 comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%. at least 99%, or 100% identical to SEQ ID NO: 9785; and the RRS-2 comprises or consists of an amino acid sequence that is al least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%. at least 99%. or 100% identical to SEQ ID NO: 9813; xxvi) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%. at least 99%. or 100% identical to SEQ ID NO: 9762; the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%. at least 99%, or 100% identical to SEQ ID NO: 9786; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%. at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9814; or xxvi i ) the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%. at least 80%. at least 85%, at least 90%, at least 95%. at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9763; the RRS-1 comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%. at least 99%. or 100% identical to SEQ ID NO: 9787; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9815.9) A system for the modification of a target nucleic acid comprising: a) a recombinase or a nucleic acid encoding a recombinase; b) a donor DNA vector comprising a first recombinase recognition sequence(RRS-1), wherein the target nucleic acid comprises a second RRS (RRS-2), wherein the recombinase comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%. at least 99%, or 100% identical to a protein any one of SEQ ID of column 3 of TABLE 16, and c) wherein at least one of the RRS-1 and RRS-2 comprises a corresponding kmer sequence of TABLE 16; wherein in the protein and the kmer are on the same line of TABLE 16.

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