Methods and compositions for integration of RNA into a DNA genome

Abstract

Described herein are methods for integrating target RNA into DNA. Also described are compositions comprising reverse transcriptase molecules which can initiate cDNA synthesis by multiple mechanism to synthesize cDNAs.

Description

[0001] Attorney Docket No.10046-551WO1 METHODS AND COMPOSITIONS FOR INTEGRATION OF RNA INTO A DNA GENOME CROSS-REFERENCE TO RELATED APPLICATIONS This application claims benefit of U.S. Provisional Application No.63 / 519,954, filed August 16, 2023, incorporated herein by reference in its entirety. SEQUENCE LISTING A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via PatentCenter encoded as XML in UTF-8 text. The electronic document, created on August 14, 2024, is entitled “10046-551WO1_ST26.xml”, and is 190,846 bytes in size. GOVERNMENT SUPPORT CLAUSE This invention was made with government support under Grant no. R35 GM136216 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND Bacteria face incessant attacks by viruses and genomic parasites and have evolved defense systems to combat these threats. Among the most widely studied are CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) systems found in diverse bacteria and archaea (1). CRISPR-Cas systems typically include an RNA-guided nuclease (effector) complex, a CRISPR repeat locus that accepts snippets of nucleic acids (protospacers) derived from threats, enzymes (Cas4, Cas5, or Cas6) that process transcribed spacers into RNA guides for nuclease effector complexes, and a DNA integrase (Cas1 / Cas2) that site-specifically integrates new spacers from invading pathogens into CRISPR arrays (2–8). Six types of CRISPR systems (Type I to VI) each with multiple subclasses have been distinguished (1). Type III systems differ from the others in their ability to cleave both DNA and RNA in a transcriptionally coupled reaction (9–11). Additionally, some Type III CRISPR systems have an associated reverse transcriptase (RT), either co- expressed with Cas1 / Cas2 or present as a reverse transcriptase-Cas1 (RT-Cas1) fusion protein, some of which also have an N-terminal Cas6 domain that processes guide RNAs for incorporation into effector complexes (12–16). Most CRISPR-associated RTs are closely related to the RTs encoded by mobile group II introns, prolific bacterial retrotransposons whose dissociated RTs have evolved in different bacteria to perform a variety of cellular Attorney Docket No.10046-551WO1 functions (17–22). Four RT-Cas1 proteins have been shown to site-specifically integrate RNA as well as DNA into CRISPR arrays in vivo (12, 23–25), but the mechanisms by which RNA protospacers are reverse transcribed and integrated into CRISPR arrays have remained unclear. Although the six types of CRISPR systems vary in the mechanisms used for processing guide RNAs and destroying invading nucleic acids, all use the same Cas1 / Cas2- mediated cleavage-ligation (transesterification) mechanism to site-specifically integrate double-stranded DNA (dsDNA) protospacers into CRISPR arrays (26–30). Structural studies have shown that Cas1 and Cas2 form a hexameric complex that binds a dsDNA protospacer across its length with overhanging or splayed single-stranded 3’ ends of opposite strands inserted in one or both active sites of Cas1 proteins on opposite sides of the complex (25, 31– 37). Cas1 / Cas2 complexes from different organisms have structural variations that favor different length spacers and splay open different length single-stranded 3’ ends (30, 38). A single-molecule study of spacer acquisition by an Enterococcus faecalis Type II-A CRISPR system indicated that the Cas1 / Cas2 complex remains stably bound to the integrated spacer until it is dislodged by transcription-coupled DNA repair, which fills in and seals single- stranded gaps to fully integrate the newly acquired spacer into the host genome (39). This process enables integration of protospacers into CRISPR arrays without introducing deleterious double-strand breaks in bacterial chromosomal DNA. Biochemical analyses of the mechanism by which RT-Cas1 proteins acquire spacers from RNA have been sparse. The 4 RT-Cas1 fusion proteins that acquire spacers from RNA are comprised of an RT domain corresponding to the fingers and palm of retroviral RTs, but fused directly to Cas1 rather than a canonical thumb domain as in other RTs (Fig.1A). The RT domain of RT-Cas1 proteins contains 7 conserved sequences blocks (RT1-7) found in all RTs plus an N-terminal extension (NTE) with an RT0 loop and two expanded regions (RT2a and RT3a) between the conserved RT sequence blocks (Fig. 1A). These additional regions are absent in retroviral RTs, but structurally conserved and functionally important in group II intron and other bacterial RTs, as well as in LINE-1 and other eukaryotic non-long terminal repeat (LTR)-retrotransposon RTs (collectively termed non-LTR-retroelement RTs) (40). The Marinomonas mediterranea (Mm) RT-Cas1 protein, associated with a Type III-A CRISPR system, was shown to function in complex with Cas2 to site-specifically integrate DNA and RNA protospacers into CRISPR arrays in vivo and in vitro (12). It was also shown to have an active N-terminal Cas6 domain that functions in CRISPR RNA processing and whose interaction with the RT domain is required for RT activity (14). A cryo-EM structure Attorney Docket No.10046-551WO1 showed that a closely related Thiomicrospira Type III Cas6-RT-Cas1 / Cas2 forms a hexameric complex similar to Cas1 / Cas2 proteins that acquire spacers from DNA, but with the Cas6 and RT domains interacting with each other to form separate flexibly attached lobes and structural differences in regions of Cas1 / Cas2 that function in protospacer binding (14, 25). The Fusicatenibacter saccharivorans (Fs) and Vibrio vulnificus (Vv) RT-Cas1 proteins, both associated with Type III-D CRISPR systems, were shown to acquire RNA-derived spacers in vivo, but have not been investigated biochemically (23, 24). Mm RT-Cas1 / Cas2 could acquire both RNA and DNA protospacers in its native host, but only DNA protospacers in E. coli, suggesting that host-specific factors contribute to RNA protospacer acquisition in vivo (12). What is needed in the art are methods and compositions which allow for site-specific integration of RNA into DNA. SUMMARY Disclosed herein is a method of integrating a target RNA into DNA, the method comprising modifying a target RNA by inserting or substituting one or more deoxynucleotide segments into the target RNA, then exposing the modified target RNA to a DNA integrase or recombinase, wherein said DNA integrase or recombinase integrates the target RNA into the DNA. Also disclosed herein is an isolated nucleic acid molecule comprising a genomic DNA with an integrated RNA target, wherein the RNA target comprises at least one or more deoxynucleotides which are not part of the genomic DNA. Further disclosed herein is a method of reverse transcribing all or part of a target RNA into a cDNA, the method comprising incubating the target RNA with a) an RT that can initiate cDNA synthesis by multiple mechanisms, wherein said multiple mechanisms comprise de novo initiation, protein primer or the use of short (<10 nt) exogenous or RT-synthesized DNA oligonucleotide protein; and b) dNTPs; wherein said incubation is carried out under conditions suitable for reverse transcription, thereby producing cDNA. Disclosed is a method of determining RNA sequences, the method comprising the steps of: a) obtaining a sample comprising two or more different RNA sequences; b) reverse transcribing the RNA sequences using an RT that is capable of using multiple mechanisms for initiating reverse transcription at different 3' proximal sites that enable cDNA synthesis from diverse RNAs in proportion to their abundance; wherein said reverse transcribing is done under conditions suitable for reverse transcription, thereby producing cDNA; c) adding Attorney Docket No.10046-551WO1 adapters to cDNA produced in step b); and d) analyzing cDNA from step c) to determine RNA sequences. Also disclosed is a method of editing target DNA using prime editing without an added primer, the method comprising: a) contacting the target DNA with a pegRNA complex and an RT that can initiate cDNA synthesis by a combination of mechanisms, including de novo initiation, protein priming, or the use of exogenous or RT-synthesized DNA oligomer primers; wherein said RT is capable of reverse transcription without an added primer, and further wherein said pegRNA complex provides a replacement sequence for the target DNA; b) incubating the target DNA, pegRNA, and RT under conditions suitable for: i) binding of pegRNA complex to one strand of target DNA; ii) nicking an opposite strand of the target DNA from where the pegRNA complex is bound using a nickase, thereby forming a flap of target DNA, wherein said flap of target DNA comprises the sequence for editing; iii) allowing the replacement sequence of pegRNA to hybridize to the flap of target DNA; iv) using RT to copy the remaining portion of the flap, thereby forming a double-stranded flap; v) removing the replacement sequence of the pegRNA so that edited target DNA hybridizes with the opposite strand of target DNA, wherein said opposite strand of target DNA now comprises a mismatch; and vi) repairing the mismatched opposite strand of target DNA so that both strands comprise edited target DNA. A composition comprising an isolated reverse transcriptase (RT), wherein said RT can initiate cDNA synthesis by multiple mechanisms, wherein said multiple mechanisms comprise de novo initiation, protein primers, or the use of short (<10 nt) exogenous or RT- synthesized DNA oligonucleotide proteins to synthesize a full-length or near-full length cDNA copy of a target RNA. DESCRIPTION OF DRAWINGS Fig.1A-D shows spacers acquired by RT-Cas1 / Cas2 proteins have higher numbers of non-coded nucleotides at spacer-repeat junctions than those acquired by Cas1 / Cas2 proteins lacking an RT domain. (A) Schematics of RT-Cas1 / Cas2 and Cas1 / Cas2 proteins associated with Type III CRISPR systems from Marinomonas mediterranea (Mm); Fusicatenibacter saccharivorans (Fs); Vibrio vulnificus (Vv); Thermus thermophilus (Tt); Staphylococcus epidermidis (Se); and Streptococcus thermophilus (St). RT1-7 (black), conserved sequence blocks present in all RTs; NTE / RT0, RT2a and RT3a (red), structurally conserved regions of non-LTR-retroelement RTs that are absent in retroviral RTs. (B) Analysis of non-coded nucleotides associated with leader-proximal spacers acquired in vivo Attorney Docket No.10046-551WO1 by different RT-Cas1 / Cas2 or Cas1 / Cas2 proteins. Left, numbers of spacers with non-coded nucleotides at their leader-proximal end only, leader-distal end only (red), both ends (brown), or neither end (none, white). Right, numbers of spacers with a single (black) or ^2 non-coded nucleotides (red), excluding small numbers of spacers with non-coded nucleotides at both spacer-repeat junctions. The analysis was done using datasets listed in Materials & Methods for unique spacer sequences mapped to the host or phage genomes (Table 4). The inset bar graphs for the smaller number of Vv RT-Cas1 / Cas2 spacers are plotted on a different scale (x100). (C) Stacked bar graphs comparing the number of spacers acquired by Mm WT RT-Cas1 / Cas2 or RT ^-Cas1 / Cas2 with A, C, G, or T residues at non- coded nucleotide positions N1 to N5. The analysis was done for spacers that mapped to the M. mediterranea strain MMB1 genome and had non-coded nucleotides at one end only, putatively the 3’ end used for terminal transferase addition of non-coded nucleotides. (D) Length distribution of unique spacer sequences acquired by Mm WT RT-Cas1 (black) or RT ^-Cas1 (red). Left, length distribution of spacers defined as the sequence between two repeats; right, length distribution of the same spacers after removing non-coded nucleotides. Fig.2A-B shows nucleotide preferences for addition of non-coded deoxynucleotides to the 3’ end of RNA or DNA oligonucleotide protospacers by Mm RT-Cas1 / Cas2 terminal transferase activity. Terminal transferase assays were done as described in Materials & Methods by incubating purified Mm WT RT-Cas1 / Cas2 with 5’32P-labeled 29-nt RNA (R29, A) or DNA (D29, B) oligonucleotides having the same nucleotide sequence and 1 mM of the indicated dNTP in reaction medium containing 10 mM MgCl2 without (-) or with (+) 1 mM MnCl2. After incubating at 37˚C for times up to 2 h, the products were analyzed in a denaturing 6% polyacrylamide gel, which was dried and quantified with a phosphorimager. Lighter exposures showing resolution of single-nucleotide extensions are appended below the bottom of gel. The hatch marks to the right of the gel indicate the positions of 5’-labeled size markers (10-nt ssDNA ladder; Invitrogen) in a parallel lane. The plots show the fraction of labeled product as a function of time with the data fit to a single exponential equation to obtain values for kobs and amplitude. R2values for curve fits are listed in Table 2. A dash indicates no detectable extension confirmed by the lighter exposure of the bottom part of the gel. A repeat of the terminal transferase assays with 5’- labeled oligonucleotides and unlabeled dNTPs incubated for 1 h at 37˚C showed similar nucleotide preferences. Fig.3.A-C shows RNA protospacer integration into a CRISPR array by Mm RT- Cas1 / Cas2 requires 3’ deoxynucleotides. (A) Mm WT RT-Cas1 / Cas2 was incubated with Attorney Docket No.10046-551WO1 29-nt DNA or RNA protospacers having the same sequence (D29 and R29, respectively, S0) and an 88-bp internally32P-labeled (stars) CRISPR DNA in the presence or absence (None) of the indicated dNTPs for 1 h at 37˚C. The products were analyzed in a 6% polyacrylamide / urea gel, which was dried and scanned with a phosphorimager. Lanes: No Protein, labeled CRISPR DNA incubated without RT-Cas1 / Cas2 or dNTPs; M, 5’-labeled 10-nt DNA ladder; arrows, integration products whose lengths increased in the presence of dNTPs. The labeled CRISPR DNA strands ran as closely spaced doublets. (B) Mm WT RT- Cas1 / Cas2 was incubated for 1 h at 37˚C with 5’32P-labeled (red asterisk) D29 and R29 oligonucleotide protospacers without or with different length 3’-dA tails and a 3’-blocked unlabeled 68-bp CRISPR DNA. Products were analyzed in a 6% polyacrylamide / urea gel. The gap in the phosphorimager scan demarcates a lighter exposure of the bottom of the gel. (C) Time courses for integration of DNA and RNA protospacers with different length 3’-dA tails (denoted dAn). Reactions were done with 5’-labeled protospacers as in (B) for times up to 1 h. The plots show the fraction of labeled oligonucleotide inserted into the top and bottom strands of the CRISPR DNA as a function of time fit to a single exponential equation to obtain values for kobsand amplitude. R2values for curve fits are listed in Table 2. The gels for the time courses in panel C and a full repeat of the experiment are shown in fig.12. Fig.4A-C shows RT-Cas1 / Cas2 synthesizes near full-length copies of RNA and DNA templates by initiating at 3’-proximal sites without an added primer. (A) DNA synthesis reactions were done by incubating WT RT-Cas1 / Cas2 (left) or RT∆-Cas1 / Cas2 (right) with 50-nt RNA or DNA oligonucleotide templates (250 nM) containing a 3’- proximal CCC sequence (R50CCC and D50CCC, respectively) without or with a 3’-ddC blocking group and32P-labeled dNTPs (20 ^M [ ^-32P]-dCTP + 500 ^M dATP, dGTP, and dTTP) in reaction medium containing 10 mM MgCl2 ^ 1 mM MnCl2 for 1 h at 25˚C. After phenol extraction, the samples were analyzed in a denaturing 20% polyacrylamide gel, which was dried and scanned with a phosphorimager. Lane M, 5’-labeled 10-nt ssDNA ladder size markers in a parallel lane, with a darker exposure of that lane appended on the left. (B) RT-Cas1 / Cas2 cDNA synthesis reactions done as in panel A with 3’-blocked 50-nt DNA and RNA templates containing a 3’ proximal CCC sequence. After clean up with a Zymo Oligo Clean and Concentrator kit, the products were incubated for 20 min at 37˚C in the absence or presence of RNase H or RNase A, followed by Protease K digestion and analysis in a non-denaturing 15% polyacrylamide gel. Lane M, 5’-labeled Low Molecular DNA Ladder size markers (New England Biolabs). (C) RT-Cas1 / Cas2 DNA synthesis Attorney Docket No.10046-551WO1 reactions with 3’-blocked DNA and RNA templates containing a 3’ proximal CCC or CGC sequence. Reactions were done and analyzed in a denaturing 20% polyacrylamide gel as in panel A. The schematics to the right of the gels in panels A-C depict the labeled cDNAs (line with stars) synthesized from RNA or DNA templates. Fig.5A-D shows TGIRT sequencing of cDNAs synthesized by Mm RT-Cas1 / Cas2 from RNA templates containing a 3’-proximal CCC or CGC sequence. (A) R50CCC_ddC; (B) and (C) R50CCC-OH; (D) R50CGC_ddC. Reverse transcription was done by incubating Mm RT-Cas1 / Cas2 (500 nM) with RNA template (250 nM) and an equimolar mix of 500 µM dATP, dCTP, dGTP and dTTP in reaction medium containing 10 mM MgCl2 without (left) or with (right) 1 mM MnCl2 for 1 h at 25˚C. After stopping the reactions by adding 25 mM EDTA, products were incubated without or with RNase A, as indicated in the Figure, followed by Protease K digestion, clean up, and construction of TGIRT-seq libraries, as described in Materials & Methods. The libraries were sequenced on an Illumina NextSeq 550 to obtain ~1 million reads for each sample. For each sample, the RNA template sequence (letters with highlights) is shown above with prevalent reads (>1% for cDNAs synthesized from 3’-blocked RNA templates and >5% for RNA templates with a 3’ OH) shown below in sequence logo format with the height of the letter corresponding to the proportion of the nucleotide at that position. Non-coded nucleotides are colored brown, and 3’-RNA template sequences used to prime snap-back cDNA synthesis are dots indicating an extended not shown RNA template sequence. Nucleotides in the RNA template corresponding to first nucleotide of a cDNA product are highlighted, with a thin line above the template extending over the first two nucleotides of the synthesized cDNA. The 3’-proximal CCC and mutant CGC sequence in the template are highlighted in red. cDNA start sites identified by sequencing were consistent with gel analysis of32P-labeled cDNAs. SEQ ID NOS can be found in Table 1 along with corresponding reference number found in figure. Fig.6A-D shows Mm RT-Cas1 / Cas2 can initiate DNA synthesis at 3’-proximal sites in RNA and DNA templates by protein-priming with any dNTP. (A) Mm RT-Cas1 / Cas2, RT-Cas1, or Cas2 (500 nM) were incubated with [ ^-32P]-dGTP (5 µCi; 83 nM) without (-) or with R50CCC_ddC and D50CCC_ddC templates (250 nM) in reaction medium containing 10 mM MgCl2 ^ 1 mM MnCl2 for 15 min at 25˚C. Reactions were chased with unlabeled dNTPs (500 µM equimolar mix) for 30 min at 25˚C, and then incubated with RNase A, MNase or Protease K (15 min at 25˚C), as indicated. Reactions with RNA (left) and DNA (right) templates were done and analyzed in parallel on SDS-polyacrylamide Attorney Docket No.10046-551WO1 gradient gels (autoradiograms, top; Coomassie blue-stained, bottom). M, Precision Plus (Bio-Rad) size markers. (B) Reactions done as in (A) by incubating RT-Cas1 / Cas2 with R50CCC_ddC template and different [ ^-32P]-dNTPs with 10 mM MgCl2 and 1 mM MnCl2. (C) Reactions done as in (B) by incubating RT-Cas1 / Cas2 with 3’-blocked RNA or DNA templates with different 3’-proximal trinucleotides and [ ^-32P]-dNTPs for 15 min followed by a 30-min chase with the same dNTP (500 µM). (D) Reactions done as in (B) by incubating RT-Cas1 / Cas2 with R50CCC_ddC template and [ ^-32P]-dGTP for 7.5 (left) or 15 min (right), followed by a 30-min chase without (-) or with dGTP (500 µM), dGTP + ddGTP (500 and 1,000 µM, respectively), or an equimolar mix of dNTPs (500 µM each). After stopping the reactions with EDTA, products were incubated for 15 min ± Protease K (0.32 units) and analyzed in a 20% polyacrylamide / urea gel. M, 5’-labeled 10-nt ssDNA ladder (left) and 5’-labeled dGMP2and dGMP3(right). Relative efficiencies of protein labeling by different dNTPs in panel C were the same as in panel B. Fig.7A-C shows Mm RT-Cas1 / Cas2 synthesizes near full-length cDNAs from different RNA templates by using multiple mechanisms to initiate cDNA synthesis at different 3’ proximal sites. (A) Gel analysis of cDNAs synthesized by Mm WT RT- Cas1 / Cas2 using exogeneous dinucleotide primers. Reactions were done by incubating RT- Cas1 / Cas2 (500 nM) with 250 nM 3’-blocked RNA templates with 3’-proximal AAA, CCC, GGG, UUU or CGC without or with 20 µM 5’-32P-labeled (*) dA2, dC2, dG2, dT2dinucleotide primers in reaction medium containing 10 mM MgCl2 without MnCl2. After incubating for 30 min at 25˚C, the reactions were chased for 30 min with 500 µM of the dNTP matching the dinucleotide primer or an equimolar mix of all 4 dNTPs (500 µM each). (B) Gel analysis of cDNAs synthesized from the same templates as in panel A without an added primer. RT-Cas1 / Cas2 was incubated with RNA templates (250 nM) and32P-labeled dNTPs (32P-dCTP, dATP, dGTP, and dTTP, 500 ^M each) for 1 h at 25˚C. (C) Gel analysis of cDNAs synthesized from 3’-blocked templates with varying length RNA segments and 3’-dA tails. Reactions were done with 500 nM RT-Cas1 / Cas2, 250 nM template and32P- labeled dNTPs (20 µM [ ^-32P]-dCTP plus 500 µM dATP, dGTP, and dTTP) in reaction medium containing 10 mM MgCl2 ± 1 mM MnCl2 for 1 h at 25˚C.32P-labeled cDNAs were analyzed in a denaturing 20% polyacrylamide gel against a 5’-labeled 10-nt ssDNA ladder (lanes M). Gel analysis for all samples was consistent with cDNA start sites identified by TGIRT-seq (Figs.5 and 8). Fig.8 shows TGIRT sequencing of cDNAs synthesized by Mm RT-Cas1 / Cas2 from different RNA templates in the presence or absence of added dinucleotide primers. (A) Attorney Docket No.10046-551WO1 TGIRT-seq of cDNAs from reverse transcription reactions using the same 3’-blocked RNA templates as in Fig.7A with unlabeled dinucleotides and an equimolar mix of all 4 unlabeled dNTPs (500 µM each). Reverse transcription reactions were done for 1 h at 25˚C in reaction medium containing 10 mM MgCl2 and cDNAs were analyzed by TGIRT-seq, as described in Materials & Methods. Nucleotides in the RNA template that correspond to the first nucleotide of a cDNA product are highlighted with black vertical lines indicating the percentage of reads beginning at that nucleotide. The 3’-proximal trinucleotide sequences in templates are underlined. The stacked bar graphs (right) show the proportions of different nucleotides at positions N1to N3of the cDNAs (color code shown below panel C at bottom left). (B) TGIRT-seq analysis of cDNAs synthesized from the same 3’-blocked templates as in Fig.7B in the absence of an added primer. Reverse transcription reactions were done with an equimolar mix of all 4 dNTPs (500 µM each) in reaction medium containing 10 mM MgCl2 ^ 1 mM MnCl2 for 1 h at 25˚C. The datasets for the R50CCC_ddC and R50CGC_ddC templates were the same as those in Fig.5. (C) TGIRT-seq of cDNAs synthesized from 3’-blocked RNA templates with different length RNA and dA-tail segments. The RNA templates were the same as those in Fig.7C with reverse transcription reactions done with an equimolar mix of all 4 dNTPs (500 µM each) in reaction medium containing 10 mM MgCl2 ^ 1 mM MnCl2 for 1 h at 25˚C. cDNA start sites for all templates were consistent with gel analysis of labeled cDNAs in Figs.4 and 7. SEQ ID NOS can be found with correlating name of sequence in Table 1. Fig.9 A-D shows time courses comparing rates of integration of 3’-dA-tailed RNA, ssDNA, and RNA-dA / DNA duplex protospacers into a CRISPR DNA. (A) Schematic of spacer integration assays using a CRISPR DNA with a 5’ leader (L), repeat (R1), and hairpin segment corresponding to the first spacer (S1). Spacer integration reactions were done by incubating 5’-labeled (*) 3’-dA-tailed RNAs (*RNA-dA) or ssDNAs (*DNA) with or without a complementary DNA strand that leaves different length 3’-dA overhangs on both strands. For two-site cleavage ligation of duplex protospacers, only the insertion of the RNA into the top strand is shown. (B) Time courses for production of the labeled 99-nt band resulting from cleavage / ligation reactions at the 5’ end of R1 on both strands for three sets of protospacers (I, II, and III) comprised of 3’-dA tailed R29 or R30CCC RNAs without or with a complementary DNA strand. Reactions were done by incubating WT RT- Cas1 / Cas2 (500 nM) and CRISPR hairpin DNA (100 nM) with a 5’32P-labeled (*) protospacer (5 nM) for up to 2 h with products analyzed in an 8% polyacrylamide / urea gel. Plots for *RNA-dA, *RNA-dA / DNA, RNA-dA / *DNA, and *DNA protospacers are color Attorney Docket No.10046-551WO1 coded as shown at the bottom of the Figure. The data were fit to a single exponential equation to calculate rates (kobs) and amplitudes (Ampl.). R2values are listed in Table 2. (C) Spacer integration by RT ^-Cas1 / Cas2 for the same sets of protospacers. (D) Spacer integration by WT RT-Cas1 / Cas2 for the same sets of protospacers at 250 nM protospacer concentration. Time courses in panels B-D were done once in parallel for each set of protospacers with conclusions based on consistent trends for RNA-dA, ssDNA or RNA- dA / DNA duplex protospacers that differed in sequence and length of 3’-DNA overhangs. Fig.10 shows possible pathways and mechanisms used for RNA spacer acquisition by RT-Cas1 / Cas2 proteins. All pathways start with RT-Cas1 / Cas2 terminal transferase activity adding short 3’-DNA tails (dNn) to host cellular or pathogen RNA fragments resulting from cleavage by RNases that leave either a 3’ OH or a 3’ phosphate that can be removed enzymatically to leave a 3’ OH. On the left are pathways in which RT-Cas1 / Cas2 uses different mechanism (de novo initiation, protein priming, or exogenous or synthesized short DNA oligomer primers) to synthesize cDNAs that remain annealed to the RNA-dN template in an RNA-dN / cDNA duplex. The duplex protospacers are then integrated into opposite strands of the CRISPR array by RT-Cas1 / Cas2 via a mechanism analogous to that used by conventional Cas1 / Cas2 proteins to integrate duplex DNA protospacers. On the right are alternative pathways in which 3’-dN-tailed RNA protospacers are integrated into the CRISPR array prior to cDNA synthesis. The integrated RNA-dN protospacers could then be used as a template for cDNA synthesis by RT-Cas1 / Cas2, resulting in an RNA- dN / cDNA duplex that becomes fully integrated into the CRISPR array by a mechanism analogous to the second step for integration of DNA duplex protospacers (horizontal arrow pointing left). Alternatively, a single-stranded RNA-dN protospacer integrated at one site could in principle be integrated into the opposite strand by a coupled integration / disintegration reaction, resulting in DNA gaps that are filled in by RT- Cas1 / Cas2 or a cellular DNA polymerase, e.g., Pol I, which has innate ability to copy RNA into DNA (60, 68)). In all pathways, protospacer integration leaves DNA segments with single-stranded gaps held together by bound RT-Cas1 / Cas2 and possibly other proteins until they are displaced by transcription-coupled DNA repair, enabling complete integration of the spacers without introducing deleterious double-stranded breaks (39). Fig.11A-D shows characteristics of spacers integrated by Mm WT RT-Cas1 / Cas2 and RT ^-Cas1 / Cas2 in vivo and additional RT-Cas1 / Cas2 terminal transferase assays. (A) Analysis of non-coded nucleotides at the 3’ end of the RNA sense strand of spacers inserted into the top or bottom strand of the CRISPR array by Mm WT RT-Cas1 / Cas2 or RT ^- Attorney Docket No.10046-551WO1 Cas1 / Cas2. The stacked bar graphs show the number of spacers acquired by Mm WT RT- Cas1 / Cas2 and RT∆-Cas1 / Cas2 with non-coded A, C, G, or T residues at positions N1 to N5 at the 3’ end of the RNA sequence. (B) Terminal transferase assays done as in Fig.2 for 1 h at 37˚C, but with unlabeled DNA or RNA oligonucleotide substrates and each of the 4 possible [ ^-32P]-dNTPs. D29 and R29 are the same 29-nt DNA and RNA oligonucleotides used for terminal transferase assays in Fig.2 (SEQ ID NOS: 50 and 60, respectively), and D34 and R34 are 34-nt RNA and DNA oligonucleotides whose sequence corresponds to a randomly chosen spacer acquired by Mm RT-Cas1 / Cas2 in vivo. The sequences of R29 and R34 and a schematic of the terminal transferase (TT) assay are shown below the gel. A repeat of the experiment using 5’ labeled oligonucleotides and unlabeled dNTPs and incubating for 1 h at 37˚C gave similar results. (C) Percentages of different non-coded dinucleotides at positions N1 and N2 at spacer-repeat junctions for spacers with non-coded nucleotides at only one end, assumed to be the RNA 3’ end. Those dinucleotides whose percentages at positions N1 and N2 were significantly higher for Mm WT RT-Cas1 / Cas2 than RT∆-Cas1 / Cas2 by chi-squared test (p<0.01) are highlighted and marked with an asterisk (*). (D) Distribution of spacer sequences acquired by WT RT-Cas1 / Cas2 and RT∆- Cas1 / Cas2 within the genes from which they originated. The analysis was done using the same spacer datasets as in Fig.1B with spacers mapped to the M. mediterranea genome. The locations of the spacer sequences were calculated as the percentile of the mid-point of the acquired sequence. Spacers derived from both coding and non-coding RNAs were used for this analysis. The plot shows a probability density function (y-axis) plotted for the relative position of each spacer within the gene to which it was mapped (x axis), with 1 and 100% corresponding to the 5’ and 3’ ends of the sense-strand RNA sequence, respectively. Figure 12A-C shows additional data for spacer acquisition assays with RNA and DNA oligonucleotides having different length 3’-dA tails. (A and B) Phosphorimager scans of gels for which time courses are shown in Fig.3C (panel A) and phosphorimager scans and plots for a repeat of those time courses (panel B). The phosphorimager scans were analyzed with ImageQuant TL software, and the data were fit to a single-exponential equation using Prism to calculate kobs and Amplitude (Ampl.) for the sum of the 2 indicated discrete bands corresponding to ligated protospacers. R2 values for the plots are listed in Table 2. Plots including the heterogenous label above the 2 discrete bands in some gels gave similar curves with higher rates and amplitudes. (C) Spacer acquisition assays for the same D29 and R29 oligonucleotides with different length dA tails analyzed in Fig.3B in the presence of 1 mM dATP to allow further extension of dA tails. A schematic of the assay Attorney Docket No.10046-551WO1 is shown to the right. Optimal integration of shorter RNA but not DNA protospacers into the CRISPR array required extension to 33 nt by Mm RT-Cas1 / Cas2 terminal transferase addition of more 3’-dA residues (bracket in gel). Reactions were done in parallel with and analyzed on the same gel as those without added dATP in Fig.3B. Figure 13A-B shows analysis of labeled cDNAs synthesized by Mm RT- Cas1 / Cas2 from different templates without an added primer. (A) cDNA synthesis from D50CCC and R50CCC templates with different 3’-blocking groups. Reactions were done by incubating Mm WT RT-Cas1 / Cas2 (500 nM) with D50CCC or R50CCC templates (250 nM) having different 3’-blocking groups (dideoxy C (ddC), phosphate (P) or an Inverted dT (INVdT)) and32P -labeled dNTPs (83 nM [ ^-32P]-dCTP plus an equimolar mix of 500 µM dATP, dCTP, dGTP, and dTTP) in reaction medium containing 10 mM MgCl2 ^ 1 mM MnCl2 for 1 h at 25˚C. The products were analyzed in a denaturing 20% polyacrylamide gel against a homemade ladder of 5’-labeled oligonucleotides in a parallel lane (M). The uniform size of the products synthesized from RNA and DNA templates with a 3’- phosphate or other 3’-blocking groups indicates that initiation of cDNA synthesis at 3’- proximal sites is not dependent upon a 3’ OH. A comparison in parallel for all 3’-blocking groups was done once with similar results for all 3’-blocking groups. (B) Gel analysis of cDNAs synthesized from R50NNN templates containing a 3’-proximal trinucleotide sequence with lower or higher dCTP concentrations in the32P -dNTP mix. Reactions were done as described in Materials & Methods by incubating RT-Cas1 / Cas2 (500 nM) with R50NNN_3’-ddC templates (250 nM) having AAA, CCC, GGG, UUU or CGC at the 3’- proximal trinucleotide site (nucleotides 46 to 48) in reaction medium containing 10 mM MgCl2 ^ 1 mM MnCl2 for 1 h at 25˚C. In the top panels, reactions were done with trace (83 nM) [α-32P]-dCTP plus 20 µM dCTP and 500 µM dATP, dGTP, and dTTP, and in the bottom panels, reactions were done with trace (83 nM) [-32P]-dCTP plus 500 µM dATP, dCTP, dGTP, and dTTP. The products were analyzed in a 20% polyacrylamide / 8 M urea gel against a 5’ labeled 10-nt ssDNA ladder. Black arrows indicate cDNAs initiated at the GGG and CGC sequences in the R50GGG and R50CGC templates at high dCTP concentration (bottom panel) but barely or not detectably at low dCTP concentration (top panel). The products were analyzed on two gels with lanes in the Figure rearranged to facilitate comparisons. The full gel for the bottom panels with all size markers is shown in Fig.7B. cDNA initiation sites in the trinucleotide sequence were confirmed by TGIRT sequencing (Fig.8B). Attorney Docket No.10046-551WO1 Figure 14A-C shows time courses for protein priming and synthesis of labeled cDNAs and short DNA oligomers synthesized by Mm RT-Cas1 / Cas2 from R50CCC_ddC template in the absence of an added primer and comparison of rates of cDNA synthesis from the same template in the absence or presence of a dinucleotide dG2 primer. (A) Time course for labeling of RT-Cas1 with [α-32P]-dGTP. Reactions were done as in Fig.6 with Mm WT RT-Cas1 / Cas2 (500 nM), R50CCC_ddC template (250 nM), and [α-32P]-dGTP (83 nM) in reaction medium containing 10 mM MgCl2 and 1 mM MnCl2 at 25˚C. Samples were taken from 0 to 90 min, and the reactions stopped with EDTA (25 mM). An aliquot of each sample was analyzed on an SDS 4-20% polyacrylamide gradient gel against Precision Plus Marker (BioRad; lane M). A phosphorimager scan of the gel is shown above, and the Coomassie blue-stained gel is shown below. Cas2 is present in too low concentrations to be visible in the Coomassie blue-stained gel. (B) Time courses for labeling of RT-Cas1 and synthesis of DNA products. Four different reactions were done as in Fig.6 with Mm WT RT-Cas1 / Cas2 (500 nM) and R50CCC_ddC template (250 nM) in reaction medium containing 10 mM MgCl2and 1 mM MnCl2at 25˚C. The 4 reactions contained [α-32P]- dGTP (83 nM), [α-32P]-dGTP (83 nM) + 500 µM dGTP, [α-32P]-dGTP (83 nM) + all 4 dNTPs (500 µM each), or α-32P]-dGTP (83 nM) + 500 µM dGTP and 1 mM ddGTP. Aliquots of the samples were incubated with or without Protease K and analyzed on a 20% polyacrylamide / 8 M urea gel. Size markers were a homemade mixture of32P -labeled oligonucleotides and dGMP2 and dGMP3 in parallel lanes (M). Labeled RT-Cas1 remained in the wells unless samples were digested with Protease K to yield smaller labeled protein fragments (arrows) or chased with a higher concentration of dNTPs. (C) Rates of cDNA synthesis from the R50CCC_ddC template with or without added dinucleotide (dG2) primer. RT-Cas1 / Cas2 (500 nM) was incubated with 3’-blocked R50CCC template (100 nM) in the presence of 20 µM [α-32P]-dCTP plus 500 µM dATP, dGTP, and dTTP for the indicated times at 25˚C in the presence or absence of 20 µM dG2 primer in reaction medium containing 10 mM MgCl2 ^ 1 mM MnCl2. The products were analyzed in a 20% polyacrylamide / 8 M urea gel (left). The plots to the right show the rate of synthesis of the ~50-nt cDNA product quantitated with ImageQuant and fit to a single exponential rate equation using Prism 10. Rates (kobs) and amplitudes (Ampl.) calculated from the curve fits are shown above the plots. R2 values for the plots are listed in Table 2. The full-time course in panels A-C were done once to confirm progressive labeling and to determine quantitative rates for multiple experiments done at fixed time points (e.g., Fig.4A, Fig.6 A and D, and Fig.7A). Attorney Docket No.10046-551WO1 Figure 15A-B shows RNase H control confirming integration of a stable RNA- dA / cDNA duplex into a CRISPR array. (A) Schematic of the experiment. cDNAs were synthesized by incubating 5’-labeled *R29+dA2 or *R29+dA2_ddC protospacers (250 nM) with RT-Cas1 / Cas2 (500 nM) and dNTPs (100 µM) in reaction medium containing 10 mM MgCl2in the presence or absence of 1 mM MnCl2(30 µM) and a dinucleotide dG2 or dT2 primer (20 µM) for 1 h at 25˚C. After clean-up with a Zymo Oligo Clean and Concentrator kit, the products were incubated with Mm RT-Cas1 / Cas2 (500 nM) and CRISPR hairpin DNA substrate (100 nM) for 1 h at 25˚C. EDTA was added to a final concentration of 25 mM, and the reaction products were treated with Protease K (0.32 units, 15 min, 37˚C), cleaned up with a Zymo Oligo Clean and Concentration kit, incubated with or without RNase H (12.5 units; New England Biolabs) for 15 min at 37˚C, and analyzed in an 8% polyacrylamide / 8 M urea gel against a 5’-labeled New England Biolabs low molecular weight DNA ladder run in a parallel lane (M). (B) Phosphorimager scans of the dried gel with different exposures for the top and bottom of the gel to better visualize the residual 5’- labeled RNA template / protospacer that was not digested by RNase H. Arrows point to reaction products that showed substantially lower labeling after RNase H treatment, indicating that they were RNA-dN / cDNA duplexes that had stably integrated into the CRISPR array as shown in the schematic. The lighter product bands for the 3’-blocked R29+dA2_ddC RNA template / protospacer, which should be unable to integrate into the CRISPR DNA, were not seen in the absence of dNTPs, suggesting that they reflect addition of 3’-dA tails required for integration to a fraction of the RNA protospacers that lacked or lost the 3’-ddC blocking group. Figure 16A-C shows courses for all32P-labeled products resulting from cleavage- ligation reactions at one site or both sites on the top and bottom strands of the CRISPR DNA in the protospacer integration assays of Fig.9B. (A-C) The three different sets (I, II, and III) of32P -labeled (*) protospacers used in the experiment are shown schematically at the top of each panel. For each set of protospacer, assays were done with a single-stranded *RNA-dAn, an *RNA-dAn / DNA duplex, an RNA-dAn / *DNA duplex, and a single- stranded *DNA. The plots for each product are symbol and color coded as shown at the bottom of the Figure, with numbers 1, 2, and 3 corresponding to the sites in the schematics (numbered black arrowheads) at which cleavage occurred to generate the product. Gray numbers in the schematics indicate the lengths of fragments that were unlabeled and thus were not visible in autoradiograms of the gels. In all cases, cleavage-ligation at the bottom- strand site (#1, triangles) occurred more rapidly than cleavage-ligation at the top-strand site Attorney Docket No.10046-551WO1 (#2, squares). For the protospacers in panels A and B, cleavage-ligation reactions at both sites (#3, black circles) occurred faster for the duplex protospacers than for the single- stranded protospacers, indicating that the duplex configuration enabled more rapid sequential ligation into both strands of the CRISPR array. Figure 17A-C shows time courses showing rates of integration of single-stranded and duplex DNA protospacers having the same sequences and overhang configurations as those for 3’-dA-tailed RNA and RNA / DNA duplex protospacers in Fig.9, B and C. Spacer- integration reactions by Mm WT-RT-Cas1 / Cas2 (panel A) and RT ^-Cas1 / Cas2 for 3 sets of all DNA protospacers (I, II, and III) with the same sequences and overhang configurations as RNA-dA and RNA-dA duplex protospacers in Fig.9. The plots show time courses for production of the labeled 99-nt product resulting from cleavage-ligation reactions at the 5’ end of R1 on both strands and are symbol and color coded as shown below panel B. The data were fit to a single exponential equation to calculate rates (kobs) and amplitudes (Ampl.). * indicates the 5’-labeled strand. R2 values for curve fits are shown in Table 2. (C) Time courses for production of all32P-labeled products resulting from cleavage-ligation reactions at single or both sites on the top and bottom strands for the same reactions as panel A. The plots for each product are symbol and color coded as shown at the bottom of the Figure, with numbers 1, 2, and 3 corresponding to the sites in the schematics (numbered black arrowheads) at which cleavage occurred to generate the product. Gray numbers in the schematics indicate the lengths of fragments that were unlabeled and thus not visible in autoradiograms of the gels. The time courses were done once in parallel with those for the three similarly configured RNA-dAn, ssDNA, and RNA-dAn / DNA duplex protospacers in Fig.9, B and C, with conclusions based on trends for RNA-dAn versus all DNA protospacers. DETAILED DESCRIPTION General Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. By “about” is meant within 10% of the value, e.g., within 9, 8, 8, 7, 6, 5, 4, 3, 2, or 1% of the value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when Attorney Docket No.10046-551WO1 values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of” and “consisting of” can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. Throughout the description and claims of this specification the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps. As used in the specification and claims, the singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof. As used herein, the terms "may," "optionally," and "may optionally" are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation "may include an excipient" is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient. "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels. By “reduce,” or “abrogate,” (used interchangeably) or other forms of the word, such as “reducing” or “reduction,” or “abrogating” or “abrogation” is meant lowering of an event or characteristic. It is understood that this is typically in relation to some standard or expected Attorney Docket No.10046-551WO1 value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. By “increase” or other forms of the word, such as “increasing,” is meant raising or elevating. It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. As used herein, by a “subject” is meant an individual. Thus, the “subject” can include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, chickens, ducks, geese, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), and birds. “Subject” can also include a mammal, such as a primate or a human. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician. “Control” refers to a sample or standard used for comparison with an experimental sample. In some embodiments, the control is a sample obtained from a healthy subject (or a plurality of healthy subjects), such as a subject or subjects not expected or known to have a particular polymorphism. In additional embodiments, the control is a historical control or standard reference value or range of values (such as a previously tested control sample or plurality of such samples), or group of samples that represent baseline or normal values. The term “hybridization” is defined as forming base pairs between complementary regions of two strands of DNA, RNA, or between DNA and RNA, thereby forming a duplex molecule, for example. Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (such as the Na+ concentration) of the hybridization buffer will determine the stringency of hybridization. Calculations regarding hybridization conditions for attaining particular degrees of stringency are discussed in Sambrook et al., (1989) Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, N.Y. (chapters 9 and 11). An “isolated” biological component (such as a nucleic acid molecule) has been substantially separated, produced apart from, or purified away from other biological components. Amino acid or nucleic acid molecules which have been “isolated” include amino acid or nucleic acid molecules purified by standard purification methods, as well as those chemically synthesized. Isolated does not require absolute purity, and can include nucleic Attorney Docket No.10046-551WO1 acid molecules that are at least 50% isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99% or even 100% isolated. A “nucleic acid” is a deoxyribonucleotide or ribonucleotide polymer, which can include analogues of natural nucleotides that hybridize to nucleic acid molecules in a manner similar to naturally occurring nucleotides. In a particular example, a nucleic acid molecule is a single stranded (ss) DNA or RNA molecule, such as a probe or primer. In another particular example, a nucleic acid molecule is a double stranded (ds) nucleic acid, such as a target nucleic acid. Examples of modified nucleic acids are those with altered backbones, such as peptide nucleic acids (PNA). The major nucleotides of DNA are deoxyadenosine 5′-triphosphate (dATP or A), deoxyguanosine 5′-triphosphate (dGTP or G), deoxycytidine 5′-triphosphate (dCTP or C) and deoxythymidine 5′-triphosphate (dTTP or T). The major nucleotides of RNA are adenosine 5′-triphosphate (ATP or A), guanosine 5′-triphosphate (GTP or G), cytidine 5′-triphosphate (CTP or C) and uridine 5′-triphosphate (UTP or U). Nucleotides include those nucleotides containing modified bases, modified sugar moieties and modified phosphate backbones, as known in the art. Examples of modified base moieties which can be used to modify nucleotides at any position on its structure include, but are not limited to: 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xanthine, acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N-6- isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, 5- methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2- carboxypropyl)uracil, and 2,6-diaminopurine. Examples of modified sugar moieties which may be used to modify nucleotides at any position on its structure include, but are not limited to: arabinose, 2-fluoroarabinose, xylose, and hexose, or a modified component of the phosphate backbone, such as phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, or a formacetal or analog thereof. Attorney Docket No.10046-551WO1 The term “complementary binding” as used herein occurs when the base of one nucleic acid molecule forms a hydrogen bond to the base of another nucleic acid molecule. Normally, the base adenine (A) is complementary to thymidine (T) and uracil (U), while cytosine (C) is complementary to guanine (G). For example, the sequence 5′-ATCG-3′ of one ssDNA molecule can bond to 3′-TAGC-5′ of another ssDNA to form a dsDNA. In this example, the sequence 5′-ATCG-3′ is the reverse complement of 3′-TAGC-5′. Nucleic acid molecules can be complementary to each other even without complete hydrogen-bonding of all bases of each molecule. For example, hybridization with a complementary nucleic acid sequence can occur under conditions of differing stringency in which a complement will bind at some but not all nucleotide positions. A “polymorphism” is a variation in a gene sequence. The polymorphisms can be those variations (DNA sequence differences, e.g., substitutions, deletions, or insertions) which are generally found between individuals or different ethnic groups and geographic locations which, while having a different sequence, produce functionally equivalent gene products. Typically, the term can also refer to variants in the sequence which can lead to gene products that are not functionally equivalent. Polymorphisms also encompass variations which can be classified as alleles and / or mutations which can produce gene products which may have an altered function. Polymorphisms also encompass variations which can be classified as alleles and / or mutations which either produce no gene product or an inactive gene product or an active gene product produced at an abnormal rate or in an inappropriate tissue or in response to an inappropriate stimulus. Alleles are the alternate forms that occur at the polymorphism. Polymorphisms can be referred to, for instance, by the nucleotide position at which the variation exists, by the change in amino acid sequence caused by the nucleotide variation, or by a change in some other characteristic of the nucleic acid molecule or protein that is linked to the variation. The identity / similarity between two or more nucleic acid sequences is expressed in terms of the identity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res.16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. Attorney Docket No.10046-551WO1 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biotechnology (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Additional information can be found at the NCBI web site. BLASTN is used to compare nucleic acid sequences. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences. Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a nucleic acid sequence that has 1166 matches when aligned with a test sequence having 1554 nucleotides is 75.0 percent identical to the test sequence (1166÷1554*100=75.0). The percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will always be an integer. In another example, a target sequence containing a 20-nucleotide region that aligns with 20 consecutive nucleotides from an identified sequence as follows contains a region that shares 75 percent sequence identity to that identified sequence (that is, 15÷20*100=75). One indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions, as described above. A “target nucleic acid molecule,” such as a “target RNA,” refers to a nucleic acid molecule whose detection, quantitation, qualitative detection, characterization, or a combination thereof, is intended. For example, the target nucleic acid molecule can be a defined region or particular portion of a nucleic acid molecule, for example a portion of a genome (such as a gene or a region of DNA or RNA containing a gene or portion thereof of interest). The nucleic acid molecule need not be in a purified form. Various other nucleic acid molecules can also be present with the target nucleic acid molecule. For example, the target Attorney Docket No.10046-551WO1 nucleic acid molecule can be a specific nucleic acid molecule (which can include RNA or DNA), for which editing is intended. In some examples, a target nucleic acid includes a viral nucleic acid molecule, or a bacterial nucleic acid molecule. Purification or isolation of the target nucleic acid molecule, if needed, can be conducted by methods known to those in the art, such as by using a commercially available purification kit or the like. The term “polypeptide” as used throughout this specification generally encompasses polymeric chains of amino acid residues linked by peptide bonds. Hence, insofar a protein is only composed of a single polypeptide chain, the terms “protein” and “polypeptide” may be used interchangeably herein to denote such a protein. The term is not limited to any minimum length of the polypeptide chain. The term may encompass naturally, recombinantly, semi- synthetically or synthetically produced polypeptides. The term also encompasses polypeptides that carry one or more co- or post-expression-type modifications of the polypeptide chain, such as, without limitation, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal Met removal, conversion of pro-enzymes or pre-hormones into active forms, etc. The term further also includes polypeptide variants or mutants which carry amino acid sequence variations vis-à- vis a corresponding native polypeptide, such as, e.g., amino acid deletions, additions and / or substitutions. The term contemplates both full-length polypeptides and polypeptide parts or fragments, e.g., naturally-occurring polypeptide parts that ensue from processing of such full- length polypeptides. A skilled person will understand that any polypeptide described herein can include one or more amino acid deletions, additions and / or substitutions (e.g., conservative substitutions), insofar such alterations preserve its activity. Each of these components is further described herein. As used herein, “functional variant” refers to peptides which peptide sequence differs from the amino acid sequence of the wild type protein, but that generally retains all the biological activity. Functional variants may also include modified peptides, fusion proteins (e.g., fused to another protein, polypeptide or the like, such as an immunoglobulin or a fragment thereof), or peptides having non-natural amino acids. Functional variants may have an extended residence time in body fluids. In certain embodiments, a variant has at least 80, 85, 90, 95, 99% of the biological activity. Preferably, a functional variant has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity. As used herein, the term “functional fragments” refers to a specific peptide that has a biological activity of interest, which peptide sequence is a part of the peptide sequence of the Attorney Docket No.10046-551WO1 reference peptide, and that can be of any length, provided the biological activity of peptide of reference is retained by said fragment. By “expression construct” or “expression cassette” is meant a nucleic acid molecule that is capable of directing transcription. An expression construct includes, at a minimum, one or more transcriptional control elements (such as promoters, enhancers or a structure functionally equivalent thereof) that direct gene expression in one or more desired cell types, tissues or organs. Additional elements, such as a transcription termination signal, may also be included. A “vector” or “construct” (sometimes referred to as a gene delivery system or gene transfer “vehicle”) refers to a macromolecule or complex of molecules comprising a polynucleotide to be delivered to a host cell, either in vitro or in vivo. A “plasmid,” a common type of a vector, is an extra-chromosomal DNA molecule separate from the chromosomal DNA that is capable of replicating independently of the chromosomal DNA. In certain cases, it is circular and double-stranded. An “origin of replication” (“ori”) or “replication origin” is a DNA sequence, e.g., in a lymphotrophic herpes virus, that when present in a plasmid in a cell is capable of maintaining linked sequences in the plasmid and / or a site at or near where DNA synthesis initiates. A “gene,” “polynucleotide,” “coding region,” “sequence,” “segment,” “fragment,” or “transgene” that “encodes” a particular protein, is a nucleic acid molecule that is transcribed and optionally also translated into a gene product, e.g., a polypeptide, in vitro or in vivo when placed under the control of appropriate regulatory sequences. The coding region may be present in either a cDNA, genomic DNA, or RNA form. When present in a DNA form, the nucleic acid molecule may be single-stranded (i.e., the sense strand) or double-stranded. The boundaries of a coding region are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxy) terminus. A gene can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. A transcription termination sequence will usually be located 3′ to the gene sequence. The term “promoter” is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene that is capable of binding RNA polymerase and initiating transcription of a downstream (3′ direction) coding sequence. It may contain genetic elements at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors, to initiate Attorney Docket No.10046-551WO1 the specific transcription of a nucleic acid sequence. The phrases “operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” mean that a promoter is in a correct functional location and / or orientation in relation to a nucleic acid sequence to control transcriptional initiation and / or expression of that sequence. The term “cell” is herein used in its broadest sense in the art and refers to a living body that is a structural unit of tissue of a multicellular organism, is surrounded by a membrane structure that isolates it from the outside, has the capability of self-replicating, and has genetic information and a mechanism for expressing it. Cells used herein may be naturally-occurring cells or artificially modified cells (e.g., fusion cells, genetically modified cells, etc.). As used herein, “expression” refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and / or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. A “fusion protein,” as used herein, refers to a protein having at least two heterologous polypeptides covalently linked in which one polypeptide comes from one protein sequence or domain and the other polypeptide comes from a second protein sequence or domain. A “DNA integrase” is an enzyme that catalyzes the integration of nucleic acid into other nucleic acid. For example, an integrase can integrate nucleic acid into another nucleic acid molecule. DNA integrases can integrate an RNA molecule which has been modified to include DNA molecules, such as by addition of dNTPs, into another DNA molecule. Examples of such molecules are given herein. Examples of integrases are given below. A “DNA recombinase” refers to an enzyme which can catalyze DNA exchange reactions between target site sequences. These reactions enable four basic functional modules: excision / insertion, inversion, translocation and cassette exchange, which have been used individually or combined in a wide range of configurations to control gene expression. Examples of recombinases include, but are not limited to, Cre recombinase, Hin recombinase, Tre recombinase and FLP recombinase. General Description As described in Example 1, the mechanisms used for reverse transcription and RNA protospacer integration by Mm RT-Cas1 / Cas2 have now been elucidated (NCBI Reference Attorney Docket No.10046-551WO1 Sequence: WP_013659858.1). Mm RT-Cas1 / Cas2 can initiate reverse transcription de novo at C residues by using a Mn2+-dependent primase activity with a strong preference for initiating at CC sequences (41). It is herein disclosed that Mm RT-Cas1 / Cas2 can also initiate reverse transcription at 3’-proximal sites by protein priming with any dNTP and by using short exogenous and synthesized DNA oligonucleotide primers with no fixed sequence requirements, as desired for an enzyme whose biological function is to acquire spacers from diverse RNAs. Mm RT-Cas1 / Cas2 can also add short 3’-DNA (3’-dN) extensions to RNA protospacers but the efficient integration of RNA protospacers into a CRISPR array requires synthesis of a cDNA to generate an RNA / cDNA duplex that is a preferred substrate for the Cas1 / Cas2 DNA integrase (41). It is disclosed herein that Mm RT-Cas1 / Cas2 can directly integrate single-stranded 3’-dN RNAs as well as 3’-dN-RNA / cDNA duplexes into a CRISPR array at rates comparable to similarly configured DNA protospacers with bioinformatic analysis showing that spacers acquired from 3’-dN-tailed RNAs comprised a high proportion of those acquired by RT-Cas1 / Cas2 proteins in vivo. Reverse Transcriptase Based on the findings described in Example 1, disclosed herein is a reverse transcriptase (RT) that can initiate cDNA synthesis by multiple mechanisms that do not require addition of a traditional primer (a complementary DNA oligomer). This RT can synthesize cDNA by a variety of techniques, which include protein priming, de novo synthesis, and using a short (less than 10 nucleotides in length) exogenous or synthesized DNA oligonucleotide primers. These mechanisms are shown in a schematic in Figure 7. Also described are methods of using this RT in a variety of methods, including genome engineering and DNA and RNA sequencing. This reverse transcriptase can be modified (engineered) or can be naturally occurring and isolated, so that it is used in a system in which it is not naturally occurring. The RT described herein can be complexed with a nucleic acid, such as a target RNA, or with both the target RNA as well as cDNA which it is synthesizing by reverse transcription. Based on conservation of structural features, the multiple mechanisms for initiation of cDNA synthesis described here are likely to be widely used by non-LTR- retroelement RTs. Importantly, the RT described herein can synthesize a full-length cDNA copy, or near- full length cDNA copy, of a target RNA. By “near full length” is meant, as compared to the RNA molecule from which the cDNA molecule is reverse transcribed, a cDNA molecule that is at least 80% of the length of the RNA. For example, the cDNA can be 80%, 85%, 90%, or Attorney Docket No.10046-551WO1 95% of the length of the RNA, or any amount in between or above these amounts. Furthermore, it can be 90% or more identical to a complement of the RNA (except, of course, equivalent deoxyribonucleotides replace ribonucleotides, so that “uracil” replaces “thymine”). One example of an RT which can carry out these multiple mechanisms of transcribing cDNA without a traditional primer includes an RT-Cas1 fusion protein. This RT-Cas1 fusion protein can be an isolated, naturally occurring RT-Cas1 fusion protein, such as the RT- Cas1 / Cas2 fusion protein found in Marinomonas mediterranea and described in Example 1. By “traditional primer” or “added primer” is meant a primer of more than 10 nucleotides in length. One of the techniques that the RT described herein can use to transcribe cDNA from RNA is the use of short nucleic acid primers. One of the multiple mechanisms the RT described herein can function is by transcribing cDNA using a short primer, such as one that is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In some examples, the primer is less than 6 nucleotides in length. Examples of this can be seen in Figure 5. These primers can be exogenous short primers, or they can be synthesized by the RT. These short primers can be synthesized de novo or by protein-priming or they can be introduced or derived from DNA fragments generated by natural processes in vivo. The RT described herein can be engineered from a naturally occurring RT to include the properties described herein. The term “reverse transcriptases” (i.e., RNA-directed DNA polymerases) refers to a group of enzymes having reverse transcriptase activity (i.e., that catalyze synthesis of DNA from an RNA template). In general, such enzymes include, but are not limited to, retroviral reverse transcriptase, retrotransposon reverse transcriptase, non- LTR-retrotransposon reverse transcriptases, chromosomally encoded bacterial reverse transcriptases, and mobile group II intron reverse transcriptases, which are found in bacteria, archaea, and eukaryotic organelles, mitochondria and chloroplasts that evolved from endosymbiotic bacteria. These reverse transcriptases can also include naturally occurring or engineered mutants, variants or derivatives thereof. A class of reverse transcriptases that is particularly suitable for use are stabilized reverse transcriptase fusion proteins of group II intron-derived reverse transcriptases. Examples of such RTs can be found in U.S. Patent 10,858,636, herein incorporated by reference in its entirety. Group II introns encode a class of RNAs known for their self-splicing reaction. Group II intron RTs have high processivity, fidelity, and strand and strand annealing activities in order to carry out their biological function. Group II intron-derived RTs also lack RNase H activity, which can be beneficial because RNase H specifically degrades the RNA Attorney Docket No.10046-551WO1 of RNA:DNA hybrids, which allows any RNA to be copied only once and can lead to reduced yields of full-length cDNA. These group II intron-derived RTs typically exhibit relatively high fidelity and high processivity. The fidelity of reverse transcription refers to the reliability of nucleotide incorporation during reverse transcription of RNA to DNA, with higher fidelity describing nucleotide copying with a low number of errors (e.g., misincorporations). Higher specificity can be provided by using longer and more specific primers, which requires the ability to carry out reverse transcription at higher temperatures, or specific binding of the RT to the 3' end of an RNA template. In the methods and compositions disclosed herein, an RT, such as a group II intron- derived RT, can be fused to a Cas1 protein that forms a complex with a Cas2 protein. This can be naturally occurring, or can be engineered using a variety of RTs and Cas1 molecules which are known in the art. The RT-Cas fusion molecule can be derived by a variety of methods known in the art and can include, for example, a stabilizer protein and / or a linker peptide. A stabilizer protein, as defined herein, is a protein forming part of the fusion protein that functions to increase the overall stability of the fusion protein. Stability includes the ability of the protein to retain its conformation and activity. In addition, the stabilizer protein preferably enhances the solubility of the fusion protein. A linker peptide can be positioned between the stabilizer protein and the thermostable reverse transcriptase, for example. By “positioned between,” it is meant that the linker peptide is connected by a chemical linkage (e.g., an amide linkage) to the N or C terminal of each of the stabilizer protein and the reverse transcriptase, as described in regard to fusion proteins herein. For example, the linker peptide can be connected through an amide linkage to the C terminal region of the stabilizer protein and the N terminal region of the thermostable reverse transcriptase. By non-cleavable, it is meant that the linker peptide is not readily susceptible to cleavage by a protease. The Cas1 molecule fused to the RT can be, for example, a naturally occurring Cas1 protein or a genetically engineered Cas1 protein. In the compositions and methods described herein, Cas1 can be used alone, or it can be part of a fusion protein RT-Cas1, or Cas1 can be associated with Cas2 such as a Cas1 / Cas2 complex. It is noted that each of these components (RT, Cas1, and Cas2) can be from different organisms, or can be engineered (non-naturally occurring). Alternatively, RT-Cas1 proteins, as well as Cas1 / Cas2 complexes, which are known in the art can be used with the methods disclosed herein. Multiple Methods of Reverse Transcription Attorney Docket No.10046-551WO1 Based on these findings, disclosed is a method of reverse transcribing a target RNA into cDNA, the method comprising incubating the target RNA with a) an RT, wherein the RT can initiate cDNA synthesis by a combination of mechanisms, including de novo initiation, protein priming, or the use of exogenous or RT-synthesized DNA oligomer primers; b) dNTPs, and c) a DNA primer of 1-6 nucleotides in length; wherein said incubation is carried out under conditions suitable for reverse transcription, thereby producing a cDNA. The DNA primer used to prime reverse transcription can be 1, 2, 3, 4, 5, or 6 nucleotides in length. Examples of this can be seen in Figure 5. These primers can be exogenous short primers, or they can be synthetic primers which are not naturally occurring or synthesized by the RT domain or the protein. These short primers can be synthesized de novo or by protein-priming or they can be introduced or derived from naturally occurring DNA fragments in a cell. Importantly, it is noted that these short primers are sufficient for priming reverse transcription of target RNA under suitable conditions. This method can be used in a multiplex assay, with different short primers priming different target RNA, or different short primers priming the same target RNA. The primers can be made of any of adenine, guanine, cytosine, or thymine. Different proportions of these bases can be used to prime transcription in different manners. Another mechanism which the RTs described herein can use to synthesize cDNA is by the use of protein priming. Examples of protein priming to create cDNA can be seen in Figure 4. Protein priming makes use of a hydroxyl group which can be provided by specific amino acid residues. These include, but are not limited to, Ser, Thr or Tyr residues. Therefore, disclosed herein is a method of reverse transcribing a target RNA into cDNA, the method comprising incubating the target RNA with: a) an RT, wherein the RT is capable of multiple mechanisms of reverse transcription, including protein priming; and b) dNTPs; wherein said incubation is carried out under conditions suitable for reverse transcription, thereby producing cDNA. In the methods described above, one of skill in the art can appreciate the conditions which are needed to transcribe RNA into cDNA. For example, such conditions can include the presence of Mn2+. Other components can also be included for the reaction to take place, such as reaction buffer, dNTPs, and DTT. The methods can be carried out in vitro, in a cell- free environment, or a cell. The methods can be used in amplification reactions or in detection reactions. Methods of determining RNA in a sample, such as by using RNA Seq, are further elucidated below. Modifying RNA with Deoxyribonucleotides Attorney Docket No.10046-551WO1 The RTs described herein can also introduce deoxyribonucleotides to an RNA molecule. For example, the RT-Cas1 has a terminal transferase activity that adds extra non- coded deoxynucleotides. This allows for the RNA molecule to be integrated into a DNA molecule. An example of this includes site-specific integration of RNA-derived protospacers into CRISPR arrays, but this method can be used for non-site specific integration or for any purpose in which one would want to introduce RNA into DNA. 1, 2, 3, 4, 5, 6, or more deoxynucleotides can be introduced to an RNA molecule. These deoxynucleotides can be any of guanine, adenine, cytosine or thymine, or can be a non-naturally occurring deoxynucleotide. The deoxynucleotide segment can comprise at least one adenine, and can optionally include two or more adenines. The deoxynucleotide segment can be added by a terminal transferase. In one example disclosed is a method of integrating target RNA into DNA, the method comprising modifying a target RNA by inserting or substituting one or more deoxynucleotide segments into to the target RNA. This can be done by an RT, as described in Example 1, although the use of RT to accomplish this is not required, as other methods for inserting DNA into RNA are known. For example, any terminal transferase can add the deoxynucleotides to the RNA, or the DNA / RNA molecule can be part of a synthesized RNA oligonucleotide (as can be seen in Figures 2C and 6). Once the target RNA has been modified with at least one deoxynucleotide, it can then be exposed to a DNA integrase or recombinase, wherein the DNA integrase or recombinase can integrate the target RNA into the DNA. Figure 7 shows a schematic of how this can occur. When more than one deoxynucleotide is inserted or substituted into the RNA molecule, they can be contiguous, or they can be non-contiguous with respect to each other. The one or more deoxynucleotides can occur at or near the 3’ end of the RNA target. For example, the deoxynucleotides can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more bases away from the 3’ end, or can be the very end. Any integrase or recombinase which can recognize the DNA or an RNA containing DNA segments can be used to integrate it into a DNA molecule such as a CRISPR array or a DNA genome. The integrase / recombinase can be one found in nature, such as those associated with various viruses, or can be orthogonal (engineered). Examples of such integrases include Cas1, Cas2, or the Cas1 / Cas2 complex. It can also include an RT-Cas1 fusion. Other such integrases include, but are not limited to, retroviral RT / integrases, or DNA integrases or recombinases of any type. The integrase can be site-specific or non-site-specific. When an RT is included in the integrase, it can be bacterial or eukaryotic in origin, and can Attorney Docket No.10046-551WO1 comprise, for example, a non-LTR-retroelement RT. The RT can be naturally occurring or can be engineered. It can be associated with a CRISPR / Cas system, or not. A DNA recombinase can also perform as an integrase and can catalyze DNA strand exchange reactions. The target RNA which can be modified with deoxynucleotides can be any length. For example, it can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides in length. It can be 20, 30, 40, 50, 60, 70, 80, 90, 100, or more nucleotides in length, or any amount between, below, or above these amounts. The DNA into which the target RNA is inserted can also be any length, as long as it is long enough to allow the integrase to stably interact with it. The DNA can be 10, 20, 30, 40, 50, 60, 70-, 80, 90, or 100 or more nucleotides in length, or any amount below, above, or in-between these values. It can be genomic DNA, for example. The target RNA with DNA segments can be inserted into a DNA location, a CRISPR complex or complex DNA genome of any organism. It can also be used for any other purpose in which it would be desired to have RNA integrated into DNA. Disclosed herein is a nucleic acid molecule comprising genomic DNA which comprises an integrated RNA target, wherein the RNA target comprises at least one or more non-naturally occurring deoxynucleotides. This modified nucleic acid, which is non- naturally occurring, can be found in a plasmid or other vector, for example, or a DNA genome. It can also be found in a CRISPR array or at any other location in a DNA genome. By “genomic DNA” is meant all or a fragment of a genome. For example, it can comprise a single gene or multiple genes, or intronic segments. It can comprise 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 or more nucleotides, or any amount in between, above, or below these values. RNA Sequencing As mentioned above, the RT molecules described herein can be used in methods of sequencing / detection. Specifically, disclosed herein is a method of carrying out RNA-Seq using the RT molecules described herein which are capable of initiating cDNA synthesis using multiple mechanisms. This can be done in a method of determining proportional amounts of RNA in a sample, the method comprising the steps of: a) obtaining a sample comprising two or more different RNA sequences; b) reverse transcribing the RNA sequences using an RT, wherein said RT does not require an added primer, wherein said reverse transcribing is done under conditions suitable for reverse transcription, thereby producing cDNA; c) adding adapters to cDNA produced in step b); and d) analyzing cDNA from step Attorney Docket No.10046-551WO1 c) to determine proportional starting amounts of RNA in the sample. The RNA sample can be a whole cell sample, or can be a tissue sample. For example, RNA sequencing of cellular RNAs can be carried out in proportion to their cellular concentrations with minimal or no sequence bias. This is advantageous for RNA-Seq applications that require comprehensive analysis of cellular RNAs. The cDNA can be analyzed using next generation sequencing (NGS). NGS libraries produced by methods of the invention are those whose nucleic acid members include a partial or complete sequencing platform adapter sequence at their termini useful for sequencing using a sequencing platform of interest. Sequencing platforms of interest include, but are not limited to, the HiSeg™, MiSeg™ and Genome Analyzer™ sequencing systems from Illumina®; the Ion PGM™ and Ion Proton™ sequencing systems from Ion Torrent™; the PACBIO RS II Sequel system from Pacific Biosciences, the SOLiD sequencing systems from Life Technologies™, the 454 GS FLX+ and GS Junior sequencing systems from Roche, the MinION™ system from Oxford Nanopore, or any other sequencing platform of interest. The RNA sample that includes the template RNA may be combined into the reaction mixture in an amount sufficient for producing the product nucleic acid. According to one embodiment, the RNA sample is combined into the reaction mixture such that the final concentration of RNA in the reaction mixture is from 1 fg / μL to 10 μg / μL, such as from 1 μg / μL to 5 μg / μL, such as from 0.001 μg / μL to 2.5 μg / μL, such as from 0.005 μg / μL to 1 μg / μL, such as from 0.01 μg / μL to 0.5 μg / μL, including from 0.1 μg / μL to 0.25 μg / μL. In certain aspects, the RNA sample that includes the template RNA is isolated from a single cell. In other aspects, the RNA sample that includes the template RNA is isolated from 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, 20 or more, 50 or more, 100 or more, or 500 or more cells, such as 750 or more cells, 1,000 or more cells, 2,000 or more cells, including 5,000 or more cells. According to certain embodiments, the RNA sample that includes the template RNA is isolated from 500 or less, 100 or less, 50 or less, 20 or less, 10 or less, 9, 8, 7, 6, 5, 4, 3, or 2 cells. The template RNA may be present in any nucleic acid sample of interest, including but not limited to, a nucleic acid sample isolated from a single cell, a plurality of cells (e.g., cultured cells), a tissue, an organ, or an organism (e.g., bacteria, yeast, or higher eukaryotic organisms, such as a plant, or a mouse, or a worm, or the like). In certain aspects, the nucleic acid sample is isolated from a cell(s), tissue, organ, and / or the like of a mammal (e.g., a human, a rodent (e.g., a mouse), or any other mammal of interest). In other aspects, the sample may be isolated from a bodily compartment suitable for use in diagnosis, such as blood, urine, Attorney Docket No.10046-551WO1 saliva, platelets, microvesicles, exosomes, serum, or other bodily fluids. In other aspects, the nucleic acid sample is isolated from a source other than a mammal, such as bacteria, yeast, insects (e.g., drosophila), amphibians (e.g., frogs (e.g., Xenopus)), viruses, plants, or any other non-mammalian nucleic acid sample source. Approaches, reagents and kits for isolating RNA from such sources are known in the art. For example, kits for isolating RNA from a source of interest—such as the NucleoSpin®, NucleoMag® and NucleoBond® RNA isolation kits by Clontech Laboratories, Inc. (Mountain View, Calif.)—are commercially available. In certain aspects, the RNA is isolated from a fixed biological sample, e.g., formalin-fixed, paraffin-embedded (FFPE) tissue. RNA from FFPE tissue may be isolated using commercially available kits—such as the NucleoSpin® FFPE RNA kits by Clontech Laboratories, Inc. (Mountain View, Calif.). Prime Editing Also described herein is a method of editing target DNA using prime editing without a primer. This method comprises: a) contacting the target DNA with a pegRNA complex and an RT, wherein said RT is capable of reverse transcription by multiple mechanisms without an added traditional primer, and further wherein said pegRNA complex provides a replacement sequence for the target DNA; b) incubating the target DNA, pegRNA, and RT- Cas1 under conditions suitable for: i) binding of pegRNA complex to one strand target DNA; ii) nicking an opposite strand of the target DNA from where the pegRNA complex is bound using a Cas protein, thereby forming a flap of target DNA, wherein said flap of target DNA comprises the sequence for editing; iii) allowing the replacement sequence of pegRNA to hybridize to the flap of target DNA; iv) using RT to copy the remaining portion of the flap, thereby forming a double-stranded flap; v) removing the replacement sequence of the pegRNA so that edited target DNA hybridizes with the opposite strand of target DNA, wherein said opposite strand of target DNA now comprises a mismatch; and vi) repairing the mismatched opposite strand of target DNA so that both strands comprise edited target DNA. Prime editing is explained in Pierre-François Perroud, “Improved prime editing allows for routine predictable gene editing in Physcomitrium patens”, Journal of Experimental Botany, 2023, herein incorporated by reference in its entirety for its teachings concerning prime editing, as well as in U.S. Patent 11,447,770, also incorporated by reference in its entirety. Vectors, Cells, and Systems Attorney Docket No.10046-551WO1 Disclosed herein are vector systems of one or more vectors, the one or more vectors comprising one or more polynucleotides encoding one or more components described herein, or combination thereof. The one or more polynucleotides in the vector systems may comprise one or more regulatory elements operably configures to express the polypeptide(s) and / or the nucleic acid component(s), optionally wherein the one or more regulatory elements comprise inducible promoters. As used herein, a “vector” is a tool that allows or facilitates the transfer of an entity from one environment to another. It is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. Generally, a vector is capable of replication when associated with the proper control elements. The term “vector” includes cloning and expression vectors, as well as viral vectors and integrating vectors. An “expression vector” is a vector that includes one or more expression control sequences, and an “expression control sequence” is a DNA sequence that controls and regulates the transcription and / or translation of another DNA sequence. Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, tobacco mosaic virus, herpes viruses, cytomegalovirus, retroviruses, vaccinia viruses, adenoviruses, and adeno-associated viruses. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, Wis.), Clontech (Palo Alto, Calif.), Stratagene (La Jolla, Calif.), and Invitrogen / Life Technologies (Carlsbad, Calif.). By way of example, some vectors used in recombinant DNA techniques allow entities, such as a segment of DNA (such as a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a target cell. The present invention comprehends recombinant vectors that may include viral vectors, bacterial vectors, protozoan vectors, DNA vectors, or recombinants thereof. With regards to recombination and cloning methods, mention is made of U.S. patent application Ser. No.10 / 815,730, the contents of which are herein incorporated by reference in their entirety. A vector may have one or more restriction endonuclease recognition sites (whether type I, II or IIs) at which the sequences may be cut in a determinable fashion without loss of an essential biological function of the vector, and into which a nucleic acid fragment may be spliced or inserted in order to bring about its replication and cloning. Vectors may also comprise one or more recombination sites that permit exchange of nucleic acid sequences between two nucleic acid molecules. Vectors may further provide primer sites, e.g., for PCR, transcriptional and / or translational initiation and / or regulation sites, recombinational signals, Attorney Docket No.10046-551WO1 replicons, selectable markers, etc. A vector may further contain one or more selectable markers suitable for use in the identification of cells transformed with the vector. Vectors capable of directing the expression of genes and / or nucleic acid sequence to which they are operatively linked, in an appropriate host cell (e.g., a prokaryotic cell, eukaryotic cell, or mammalian cell), are referred to herein as “expression vectors.” If translation of the desired nucleic acid sequence is required, the vector also typically may comprise sequences required for proper translation of the nucleotide sequence. The term “expression” as used herein with regards to expression vectors, refers to the biosynthesis of a nucleic acid sequence product, i.e., to the transcription and / or translation of a nucleotide sequence. Expression also refers to biosynthesis of a microRNA or RNAi molecule, which refers to expression and transcription of an RNAi agent such as siRNA, shRNA, and antisense DNA, that do not require translation to polypeptide sequences. In general, expression vectors of utility in the methods of generating and compositions which may comprise polypeptides of the invention described herein are often in the form of “plasmids,” which refer to circular double-stranded DNA loops which, in their vector form, are not bound to a chromosome. In some embodiments of the aspects described herein, all components of a given polypeptide may be encoded in a single vector. For example, in some embodiments, a vector may be constructed that contains or may comprise all components necessary for a functional polypeptide as described herein. In some embodiments, individual components (e.g., one or more monomer units and one or more effector domains) may be separately encoded in different vectors and introduced into one or more cells separately. Moreover, any vector described herein may itself comprise predetermined Cas and / or component polypeptides encoding component sequences, such as an effector domain and / or other polypeptides, at any location or combination of locations, such as 5′ to, 3′ to, or both 5′ and 3′ to the exogenous nucleic acid molecule which may comprise one or more component Cas and / or component polypeptides encoding sequences to be cloned in. Such expression vectors are termed herein as which may comprise “backbone sequences.” Several embodiments of the invention relate to vectors that include but are not limited to plasmids, episomes, bacteriophages, or viral vectors, and such vectors may integrate into a host cell's genome or replicate autonomously in the particular cellular system used. In some embodiments of the compositions and methods described herein, the vector used is an episomal vector, i.e., a nucleic acid capable of extra-chromosomal replication and may include sequences from bacteria, viruses or phages. Other embodiments of the invention relate to vectors derived from bacterial plasmids, bacteriophages, yeast episomes, yeast Attorney Docket No.10046-551WO1 chromosomal elements, and viruses, vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, cosmids and phagemids. In some embodiments, a vector may be a plasmid, bacteriophage, bacterial artificial chromosome (BAC) or yeast artificial chromosome (YAC). A vector may be a single- or double-stranded DNA, RNA, or phage vector. Certain embodiments of the invention may relate to the use of prokaryotic vectors and variants and derivatives thereof. Other embodiments of the invention may relate to the use of eukaryotic expression vectors. The vectors which may comprise nucleic acid sequences encoding the Cas and / or component polypeptides described herein may be “introduced” into cells as polynucleotides, preferably DNA, by techniques well known in the art for introducing DNA and RNA into cells. The term “transduction” refers to any method whereby a nucleic acid sequence is introduced into a cell, e.g., by transfection, lipofection, electroporation (methods whereby an instrument is used to create micro-sized holes transiently in the plasma membrane of cells under an electric discharge, see, e.g., Banerjee et al., Med. Chem.42:4292-99 (1999); Godbey et al., Gene Ther. 6:1380-88 (1999); Kichler et al., Gene Ther.5:855-60 (1998); Birchaa et al., J. Pharm.183:195-207 (1999)), biolistics, passive uptake, lipid:nucleic acid complexes, viral vector transduction, injection, contacting with naked DNA, gene gun (whereby the nucleic acid is coupled to a nanoparticle of an inert solid (commonly gold) which is then “shot” directly into the target cell's nucleus), calcium phosphate, DEAE dextran, lipofectin, lipofectamine, DIMIRIE C, Superfect, and Effectin (Qiagen), unifectin, maxifectin, DOTMA, DOGS (Transfectam; dioctadecylamidoglycylspermine), DOPE (1,2-dioleoyl-sn- glycero-3-phosphoethanolamine), DOTAP (1,2-dioleoyl-3-trimethylammonium propane), DDAB (dimethyl dioctadecylammonium bromide), DHDEAB (N,N-di-n-hexadecyl-N,N- dihydroxyethyl ammonium bromide), HDEAB (N-n-hexadecyl-N,N- dihydroxyethylammonium bromide), polybrene, poly(ethylenimine) (PEI), sono-poration (transfection via the application of sonic forces to cells), optical transfection (methods whereby a tiny (˜1 μm diameter) hole is transiently generated in the plasma membrane of a cell using a highly focused laser), magnetofection (refers to a transfection method, that uses magnetic force to deliver exogenous nucleic acids coupled to magnetic nanoparticles into target cells), impalefection (carried out by impaling cells by elongated nanostructures, such as carbon nanofibers or silicon nanowires which were coupled to exogenous nucleic acids), and the like. Attorney Docket No.10046-551WO1 The RT, or RT-Cas1 fusion, described herein can be introduced into a cell using any method known to one of skill in the art. The term “transformation” as used herein refers to the introduction of genetic material (e.g., a vector which may comprise a nucleic acid sequence encoding a Cas and / or component polypeptides) into a cell, tissue or organism. Transformation of a cell may be stable or transient. The term “transient transformation” or “transiently transformed” refers to the introduction of one or more transgenes into a cell in the absence of integration of the transgene into the host cell's genome. Transient transformation may be detected by, for example, enzyme-linked immunosorbent assay (ELISA), which detects the presence of a polypeptide encoded by one or more of the transgenes. For example, a nucleic acid sequence encoding Cas and / or component polypeptides may further comprise a constitutive promoter operably linked to a second output product, such as a reporter protein. Expression of that reporter protein indicates that a cell has been transformed or transfected with the nucleic acid sequence encoding RT, Cas and / or component polypeptides. Alternatively, or in combination, transient transformation may be detected by detecting the activity of the RT, Cas and / or component polypeptides. The term “transient transformant” refers to a cell which has transiently incorporated one or more transgenes. In contrast, the term “stable transformation” or “stably transformed” refers to the introduction and integration of one or more transgenes into the genome of a cell or cellular system, preferably resulting in chromosomal integration and stable heritability through meiosis. Stable transformation of a cell may be detected by Southern blot hybridization of genomic DNA of the cell with nucleic acid sequences, which are capable of binding to one or more of the transgenes. Alternatively, stable transformation of a cell may also be detected by the polymerase chain reaction of genomic DNA of the cell to amplify transgene sequences. The term “stable transformant” refers to a cell, which has stably integrated one or more transgenes into the genomic DNA. Thus, a stable transformant is distinguished from a transient transformant in that, whereas genomic DNA from the stable transformant contains one or more transgenes, genomic DNA from the transient transformant does not contain a transgene. Transformation also includes introduction of genetic material into plant cells in the form of plant viral vectors involving epichromosomal replication and gene expression, which may exhibit variable properties with respect to meiotic stability. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. Attorney Docket No.10046-551WO1 For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable biomarker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable biomarker may be introduced into a host cell on the same vector as that encoding Cas and / or component polypeptides or may be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid may be identified by drug selection (e.g., cells that have incorporated the selectable biomarker gene survive, while the other cells die). With regards to transformation, mention is made to U.S. Pat. No.6,620,986, the contents of which are incorporated by reference herein in their entirety. EXAMPLES Example 1: Mechanisms used for cDNA synthesis and site-specific integration of RNA into DNA genomes by a reverse transcriptase-Cas1 fusion protein Summary Reverse transcriptase-Cas1 (RT-Cas1) fusion proteins found in some CRISPR systems enable spacer acquisition from both RNA and DNA, but the mechanism of RNA spacer acquisition has remained unclear. Here, Marinomonas mediterranea RT-Cas1 / Cas2 was found to add short 3’-DNA (dN) tails to RNA protospacers enabling their direct integration into CRISPR arrays as 3’-dN-RNAs or 3’-dN-RNA / cDNA duplexes at rates comparable to similarly configured DNAs. Reverse transcription of RNA protospacers is initiated at 3’ proximal sites by multiple mechanisms, including recently described de novo initiation, protein priming with any dNTP, and use of short exogenous or synthesized DNA oligomer primers, enabling synthesis of near full-length cDNAs of diverse RNAs without fixed sequence requirements. The integration of 3’-dN-RNAs or single-stranded (ss) DNAs is favored over duplexes at higher protospacer concentrations, which can be relevant to spacer acquisition from abundant pathogen RNAs or ssDNA fragments generated by phage-defense nucleases. These findings reveal novel mechanisms for site-specifically integrating RNA into DNA genomes with potential biotechnological applications. Results Attorney Docket No.10046-551WO1 Spacers acquired from RNA have higher numbers of non-coded nucleotides at spacer- repeat junctions than spacers acquired from DNA It was postulated whether spacers acquired from RNA might have distinctive features that could provide clues about their acquisition mechanism. To identify such features, the sequences of newly acquired spacers (i.e., those closest to the leader) for three RT-Cas1 proteins (Mm, Fs, and Vv) were compared in host strains that support spacer acquisition from RNA compared to spacers acquired from DNA by an Mm RT-Cas1 mutant lacking the RT domain (Cas6-RT ^-Cas1, denoted RT ^-Cas1) and Cas1 proteins from Thermus thermophilus (Tt), Staphylococcus epidermidis (Se), and Streptococcus thermophilus (St) Type III systems that lack an associated RT (28, 42, 43). The leader-proximal spacers acquired by these RT-Cas1 / Cas2 and Cas1 / Cas2 proteins included different proportions with non-coded nucleotides at one or both spacer-repeat junctions that did not correlate with their ability to acquire spacers from RNA (Fig.1B, left). However, the number of non-coded nucleotides at the spacer-repeat junctions differed markedly for proteins that could acquire spacers from RNA compared to those that could not. The majority of newly acquired spacers for the wild-type Mm, Fs, and Vv RT-Cas1 proteins, which acquire spacers from RNA, had two or more non-coded nucleotides at the spacer-repeat junctions, while deletion of the RT domain of Mm RT ^-Cas1 sharply decreased the proportion of spacers that had ^2 non-coded nucleotides, approaching those for the Tt, Se, and St Cas1 / Cas2 proteins, which acquire spacers from DNA (Fig.1B, right). For wild-type (WT) Mm RT-Cas1, the first two non-coded nucleotides at the spacer-repeat junction of spacers that had non-coded nucleotides at one end only, putatively the 3’ end, were predominantly A residues (>50%) followed by G >T > C residues, while spacers acquired by Mm RT ^-Cas1 / Cas2 showed less bias for A residues and had higher proportions of C and T residues at all positions (Fig.1C; confirmed by analyzing non-coded nucleotides at the 3’ end of RNA sense-strand sequences in the top and bottom strands of the CRISPR array; fig.11A). The length distribution of spacers acquired in vivo by Mm WT RT-Cas1 / Cas2 and RT ^- Cas1 / Cas2 were similar with peaks at 34-35 nucleotides (nt) (Fig.1D, left). However, spacers acquired by WT RT-Cas1 / Cas2 required addition of 1 to 6 non-coded nucleotides to achieve a length distribution similar to that of spacers acquired from DNA by RT ^-Cas1 (Fig. 1D, right). Collectively these findings suggested that Mm RT-Cas1 might have a terminal transferase activity that adds non-coded nucleotides, preferentially A residues, to the 3’ ends of RNA protospacers. Attorney Docket No.10046-551WO1 Prompted by these findings, terminal transferase activity of WT Mm RT-Cas1 / Cas2 was assayed with 29-nt RNA and DNA oligonucleotide substrates (R29 and D29, respectively) in reaction medium containing 10 mM Mg2+in the absence or presence of 1 mM Mn2+, a physiologically relevant divalent cation that modulates the activity of many polymerases (44, 45). Consistent with the in vivo findings, the assays showed that WT RT- Cas1 / Cas2 has a terminal transferase activity that adds non-coded DNA tails to the 3’ end of RNA and DNA substrates with nucleotide preferences A >> G > C > T for the RNA substrate and A > C > G >T for the DNA substrate and Mn2+strongly increasing both the activity and preference for adding A residues to the 3’ end of the RNA substrate (Fig. 2). Additional terminal transferase assays with RNA and DNA oligonucleotide substrates having a different sequence and 3’ nucleotide showed a strong bias for purines over pyrimidines but little or no bias for dA over dG residues in the presence of Mn2+(fig. 11B). Notably, the Mm RT- Cas1 / Cas2 terminal transferase had a propensity to stop or pause after addition of short (~4 nt) dN tails, particularly evident for addition of dA tails to the RNA substrate (Fig.2 and fig. 11B), possibly a feature that helps keep overall protospacer length in a range that can be accommodated by Cas1 / Cas2. The greater preference of Mm RT-Cas1 / Cas2 for adding A residues to the 3’ end of the RNA than the DNA substrate explains why spacers acquired by Mm RT-Cas1 / Cas2 in vivo have a higher proportion of A residues at RNA proximal positions 1 and 2, but lower proportions of A residues at more distal positions 3 to 5, where nucleotide addition occurs to progressively longer stretches of DNA resulting from prior dNTP additions. Further analysis showed that spacers acquired in vivo by WT Mm RT-Cas1 / Cas2 had significantly higher frequencies of non-coded AA, AG, and GG dinucleotides at non- coded nucleotide positions 1 and 2 than those acquired by Mm RT ^-Cas1 / Cas2 (fig. 11C). Despite differences in the numbers of non-coded nucleotides, the spacers acquired by both proteins were uniformly distributed throughout the gene from which they were derived (fig. 11D). Considered together, these findings suggested that addition of deoxynucleotides to the 3’ end of RNA fragment protospacers by RT-Cas1 / Cas2 terminal transferase activity might be required for RNA protospacer acquisition. Addition of deoxynucleotides to the 3' end of RNA protospacers is necessary and sufficient for RNA spacer acquisition To investigate if addition of 3’-dN tails is required for RNA spacer acquisition, spacer acquisition assays were performed with Mm WT RT-Cas1 / Cas2 in the presence or absence of dNTPs. In an initial assay, Mm RT-Cas1 / Cas2 was incubated with an internally32P-labeled double-stranded CRISPR DNA and 29-nt ssDNA (D29) or RNA (R29) protospacers having Attorney Docket No.10046-551WO1 the same nucleotide sequence in the presence or absence of each of the 4 dNTPs, dideoxy ATP (ddATP), a non-hydrolyzable dATP analog (dApCpp), or ATP. The reactions were done in the absence of Mn2+to limit terminal transferase addition of non-coded nucleotides to the 3’ ends of the labeled CRISPR DNA, and the products were analyzed in a denaturing 6% polyacrylamide gel. Spacer ligation to the 5’ end of the first repeat (R1) on opposite strands was expected to occur via transesterification reactions that yield labeled top-strand products corresponding to the cleaved leader (L, 40 nt) and the 29-nt protospacer (S0) linked to the 5’ end of R1 (S0+R1+S1, 77 nt) and labeled bottom-strand products corresponding to L+R1+S0 (104 nt) plus unlabeled S1 (13 nt, run off the gel; schematic Fig.3A). The results showed that Mm RT-Cas1 / Cas2 ligates DNA protospacers to the 5’ end of R1 on both strands in the presence or absence of added dNTPs, as expected, while efficient RNA protospacer ligation occurred only in the presence of dNTPs or dATP (Fig. 3A), the nucleotide used most efficiently by Mm RT-Cas1 / Cas2 terminal transferase activity in the absence of Mn2+(Fig.2). The DNA protospacers integrated in the presence of dNTPs, dATP, or dGTP and the RNA protospacers spacers integrated in the presence of dNTPs or dATP were slightly longer than the initial protospacers as expected for terminal transferase addition of deoxynucleotide tails prior to integration (Fig.3A, arrows in gel). The finding that dATP was by itself sufficient for RNA protospacer integration in the absence of other dNTPs indicates that RNA protospacer acquisition required only the addition of a 3’-dA tail to an RNA protospacer and not cDNA synthesis to generate a complementary cDNA strand, in agreement with a previous finding (12). To investigate how many non-coded 3’ deoxynucleotides are required for RNA spacer acquisition, spacer acquisition assays were performed with the same 29-nt DNA and RNA oligonucleotides without or with 1 to 6 dA residues added to their 3’ ends (Fig. 3B). The oligonucleotides were 5’-32P-labeled and used to assay spacer ligation into an unlabeled CRISPR DNA in the absence of added dNTPs or dATP. The oligonucleotides comprising the CRISPR array had a 3’ biotin-blocking group to prevent terminal transferase addition of dNTPs to the 3’ DNA ends. As expected, the 5’-labeled ssDNA oligonucleotides with or without a 3’-dA tail were efficiently ligated into the CRISPR DNA and produced the two expected ligation products (S0+R1+L, 77 nt and L+R1+S0, 84 nt), whose lengths increased progressively with increasing length of the dA tail (Fig. 3B, left side). By contrast, the 29-nt RNA oligonucleotide without a 3’-dA residue was not integrated into the CRISPR DNA and the RNA oligonucleotide with a single 3’-dA residue was integrated inefficiently, whereas RNA Attorney Docket No.10046-551WO1 oligonucleotides with 2 or more dA residues at their 3’ ends were used more efficiently as protospacers (Fig.3B, right side). Time courses comparing rates of integration (kobs) of the 5’-labeled 29-nt DNA or RNA protospacers with dA tails ranging in length from 2 to 6 nt showed relatively small differences for DNA protospacers but greater dependence on dA-tail length for RNA protospacers, with RNA protospacers having 4- to 6-nt dA tails integrating at rates close to those for DNA protospacers of the same length (Fig.3C and fig.12, A and B). Addition of dATP to integration reactions with the 5’-labeled R29 and D29 oligonucleotides that had different length 3’-dA tails showed that optimal integration of shorter RNA but not DNA protospacers required extension to 33 nt (fig.12C), matching the length distribution of spacers acquired by Mm WT RT-Cas1 / Cas2 in vivo (Fig. 1D). Collectively, these findings showed that addition of short 3’-dN tails by Mm RT-Cas1 / Cas2 terminal transferase activity enabled relatively efficient integration of RNA protospacers into a CRISPR array without a requirement for synthesis of a complementary DNA. RT-Cas1 / Cas2 synthesizes near full-length DNA copies of 50-nt RNA or DNA templates without an added primer To investigate how Mm RT-Cas1 / Cas2 synthesizes DNA copies of RNAs or single- stranded (ss) DNAs, 50-nt RNA or DNA oligonucleotide templates of the same sequence were used that fortuitously included a 3’-proximal CCC sequence that turned out to be a preferred cDNA initiation site for Mm RT-Cas1 / Cas2 (templates denoted R50CCC and D50CCC, respectively; Figs.4 and 5). The RNA and DNA templates were tested without or with a 3’-ddC blocking group, which prevents 3’-deoxynucleotide addition by RT-Cas1 terminal transferase activity as well as “snap-back DNA synthesis”, a reaction in which the 3’ end of a DNA or RNA template folds back to prime DNA synthesis by base pairing to short complementary sequences upstream in the same template (46). For initial experiments, DNA synthesis reactions were done by incubating the R50CCC and D50CCC templates with Mm WT RT-Cas1 / Cas2 and32P-dNTPs (a mixture of 20 ^M [ ^-32P]-dCTP + 500 ^M dATP, dGTP and dTTP) in reaction medium containing 10 mM Mg2+in the absence or presence of 1 mM Mn2+, and the products were analyzed in a denaturing 20% polyacrylamide gel. In the absence of a 3’-blocking group, RT-Cas1 / Cas2 gave two major products with both the RNA and DNA template, one of ~100 nt, the size expected for a snap-back DNA synthesis product, and the other of ~50 nt, whose synthesis was stimulated by Mn2+and corresponded to a near full-length DNA copy of the RNA or DNA template beginning near its 3’ end (Fig. 4A). As expected, the ~50-nt DNA product but not the snap-back DNA Attorney Docket No.10046-551WO1 synthesis product was seen with the 3’-blocked R50CCC_ddC and D50CCC_ddC templates and neither product was seen with Mm RT ^-Cas1 / Cas2, which lacks RT and DNA polymerase activity (Fig.4A). Analysis of the products on a non-denaturing polyacrylamide gel showed that incubation with RNase H or RNase A (lanes labeled H and A, respectively) had no effect on the electrophoretic mobility of the dsDNA resulting from copying of the DNA template, but increased the mobility of the product synthesized from the RNA template, indicating that it was a stable RNA-cDNA heteroduplex (Fig.4B). To identify cDNA start sites, the cDNAs synthesized from the 50-nt RNA template were sequenced with or without a 3’-blocking group by using a Thermostable Group II Intron Reverse Transcriptase (TGIRT)-based DNA sequencing method (Fig. 5). The cDNA synthesis reactions were done with 500 ^M of all 4 unlabeled dNTPs in the absence or presence of Mn2+. The sequences showed that >90% of the products synthesized from the 3’- blocked R50CCC_ddC template in the absence or presence of Mn2+were near full-length cDNAs that began at the CCC sequence near the 3’ end of the RNA (Fig.5A; cDNA initiation sites; 3’ CCC sequence highlighted). The remaining products began at 3’-proximal A or U residues, with the number of initiation sites increasing in the presence of Mn2+(Fig. 5A). Most but not all of the cDNAs extended to the 5’ end of the 50-nt RNA template (indicated by proportionately smaller letters in WebLogo format) and ended with non-coded TA residues. The latter reflect non-templated nucleotide addition by RT-Cas1 / Cas2 upon reaching the 3’ end of the template, an activity found for group II intron RTs, as well as other RTs and DNA polymerases (47, 48). To test if the lack of a 3’ hydroxyl (OH) in a 3’-blocked RNA template affects the use of cDNA initiation sites, DNA synthesis reactions were carried out with Mm RT-Cas1 / Cas2 and the R50CCC template without a 3’-blocking group (R50CCC_OH) and the products were sequenced without or with treatment with RNase A to degrade 3’ segments of the RNA template used to prime snap-back DNA synthesis (Fig. 5, B and C, respectively). Without RNase treatment, the major snap-back DNA synthesis initiation site identified as beginning with a sequence corresponding to the 3’ end of the RNA template was a 3’-proximal UU complementary to the 3’ terminal AA of the RNA template (Fig.5B). Surprisingly, snap-back DNA synthesis initiation sites also included the 3’-terminal AA of the RNA template, with cDNAs initiated at that site preceded by 4 or 5 non-coded nucleotides (brown), likely added by RT-Cas1 terminal transferase activity to give the 3’ end of the RNA template sufficient flexibility to snap-back and anneal to the complementary nucleotides at the 3’ end of the template (Fig. 5B, left). Most of the snap-back products with attached RNA sequences Attorney Docket No.10046-551WO1 terminated prior to reaching the 5’ end of the RNA template, possibly reflecting limited processivity of RT-Cas1 / Cas2 (Fig.5B). Sequencing of the products from the same reactions with the R50CCC-OH template after RNase treatment showed that most (~70%) of the cDNAs began at the same 3’-proximal CCC sequence found for the 3’-blocked RNA template (Fig.5C). The remainder of the RNase-treated products began at 3’-proximal A or U residues, including a substantial proportion at the major UU snap-back DNA synthesis site upstream of the 3’ CCC (see above) with most extending to the 5’ end of the template followed by non- coded TA residues (Fig. 5C). These findings indicated that aside from enabling snap-back DNA synthesis, a 3’ OH instead of a 3’-ddC blocking group did not appreciably affect Mm RT-Cas1 / Cas2 cDNA initiation sites. The ability of RT-Cas1 / Cas2 to initiate at 3’-proximal sites irrespective of the 3’ moiety was further supported by gel analysis of cDNAs synthesized from the same RNA template with a 3’-phosphate or inverted dT residue 3’-blocking group (fig.13A), a desirable characteristic enabling synthesis of near full-length cDNAs from RNA fragments generated by cellular RNases that leave different 3’ moieties. Finally, to test the requirement for a dinucleotide CC sequence, which was suggested to be a highly preferred initiation site for a Mn2+-dependent primase activity of Mm RT- Cas1 / Cas2 (41), the efficiency of cDNA synthesis from the 3’-blocked RNA and DNA oligonucleotide templates was compared in which 3’-proximal CCC initiation site was changed to CGC. Gel electrophoresis of labeled cDNAs synthesized from these templates with32P-labeled dNTPs (20 ^M32P-dCTP + 500 ^M dATP, dGTP, and dTTP) showed that this single nucleotide mutation strongly decreased production of near full-length cDNAs initiated at the trinucleotide site in the absence or presence of Mn2+(Fig. 4C). However, sequencing of the products synthesized from the same RNA templates with 500 ^M of each unlabeled dNTP showed that the CGC sequence remained a favored cDNA initiation site (79- 92% of sequenced products) in the absence or presence of Mn2+(Fig. 5D). These findings suggested that efficient initiation of cDNA synthesis at the CGC sequence might be dependent upon dCTP concentration. Confirming this dependence, gel analysis of32P-labeled cDNAs synthesized from templates having different 3’-proximal trinucleotide sequences in place of CCC showed more efficient initiation at CGC as well as at GGG at the higher dCTP concentration (fig. 13B). These experiments showed that Mm RT-Cas1 / Cas2 could initiate cDNA synthesis at different 3’-proximal sites in the absence or presence of Mn2+without an added primer or a strict requirement for a dinucleotide CC initiation site. Attorney Docket No.10046-551WO1 RT-Cas1 / Cas2 initiates cDNA synthesis at 3’-proximal sites by protein-priming with different initiating dNTPs The finding that Mm RT-Cas1 and a variety of other RTs can synthesize cDNAs that retained a 5’-triphosphate, a key indicator of de novo initiation, with a strong preference for initiation at CC sequences was based largely on experiments using short (7-20 nt) RNA substrates with limited sequence diversity (41). De novo initiation of cDNA synthesis at the CC of a 3’-tRNA-like structure was shown previously for a mitochondrial retroplasmid RT (49). However, the more diverse sequences of spacers acquired by Mm RT-Cas1 / Cas2 in vivo led us to consider an alternate mechanism based on findings that bacterial AbiK and Abi-P2 RTs function in abortive phage infection (abi) by using non-templated protein-priming to synthesize long “random” sequence ssDNAs that contribute to altruistic cell death (18, 20). Protein priming of cDNA synthesis using an OH group of a tyrosine, threonine or serine residue resulting in covalent attachment of labeled nucleotides to the protein is a well- characterized mechanism for the initiation of cDNA synthesis by a number of viral and cellular RTs (50–52). To investigate if protein-priming could be used to initiate cDNA synthesis at the 3’- proximal CCC initiation site, Mm WT RT-Cas1 / Cas2 was incubated without or with the 3’- blocked R50CCC_ddC template and [ ^-32P]-dGTP in the presence or absence of Mn2+. After the incubation, the protein was analyzed by Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and autoradiography to detect covalently bound32P-dGTP as expected for protein-priming. As shown in Fig. 6A, incubation of Mm RT-Cas1 / Cas2 with [ ^-32P]-dGTP for 15 min in the presence but not absence of the RNA template resulted in strong Mn2+-dependent labeling of RT-Cas1 as well as more weakly labeled low molecular weight bands that results below suggested were32P-dG oligomers (Fig.6A, lanes 1-4). Time courses showed that labeling of Mm RT-Cas1 by [ ^-32P]-dGTP increased progressively for times up to 60 min (fig. 14A). Adding an equimolar mixture of all 4 unlabeled dNTPs as a chase after the initial 15-min labeling period with [ ^-32P]-dGTP resulted in the appearance of a higher molecular weight labeled band that migrated above the major Coomassie-blue stained protein band, as well as increased intensity of the lower molecular weight bands (Fig. 6A, lane 5). These additional bands were insensitive to digestion with RNase A, but sensitive to digestion with Micrococcal Nuclease (MNase), indicating that they were labeled cDNA products (lanes 6, 7). Digestion with Protease K shifted the major32P-labeled protein band to a lower molecular weight and also resulted in disappearance of the labeled higher molecular weight MNase-sensitive band, suggesting that it was an extended cDNA associated with a Attorney Docket No.10046-551WO1 fraction of the protein that was below the limit of detection by Coomassie-blue staining (Fig. 6A, lane 8). The labeling of RT-Cas1 was not dependent upon the presence of Cas2, and incubating Cas2 by itself under the same conditions did not result in labeled protein (Fig.6A, lanes 9, 10). The same experiment with a 3’-blocked 50-nt D50CCC_ddC template of the same sequence gave similar results, but with somewhat lower protein labeling intensity compared to that with the R50CCC_ddC template assayed in parallel (Fig.6A, lanes 11-21). Collectively, these findings show that Mm RT-Cas1 / Cas2 can initiate DNA synthesis on RNA or DNA templates via Mn2+-stimulated protein priming. To test if protein-priming by RT-Cas1 / Cas2 was restricted to using dGTP as the initiating nucleotide, a similar experiment in which RT-Cas1 / Cas2 was incubated with the R50CCC_ddC template was carried out in the presence of each of the 4 [ ^-32P]-dNTPs (Fig. 6B). Analysis of the products on an SDS-polyacrylamide gel showed that RT-Cas1 was labeled by all 4 [ ^-32P]-dNTPs with relative efficiency dGTP > dATP > dTTP > dCTP, and in each case, chasing with an equimolar mixture of unlabeled dNTPs (dN) resulted in higher and lower molecular weight bands corresponding to labeled DNA products that were degraded by MNase but not RNase A (Fig.6B). These findings indicated that RT-Cas1 / Cas2 could initiate DNA synthesis by protein priming with any dNTP, but with dGTP favored over dATP and purines favored over pyrimidines for initiation of DNA synthesis on the 3’-blocked R50CCC template. To determine if a 3’-proximal CCC sequence is essential for protein priming, protein labeling using 3’-blocked 50-nt RNA or DNA templates of otherwise identical sequence in which the 3’-proximal CCC was changed to UUU / TTT or GGG (Fig. 6C) were compated. As in the previous experiment, RT-Cas1 was labeled by each of these dNTPs with efficiency dGTP > dATP > dCTP (Fig.6C). However, chasing with the same unlabeled dNTP resulted in a smear of dissociated32P-labeled DNA products extending up the gel lanes for RNA or DNA templates that had a complementary 3’-trinucleotide sequence but not for those that had a non-complementary trinucleotide, as expected for reiterative copying of the trinucleotide in a sequence-dependent manner (Fig.6C and see below). To confirm that protein-priming could give rise to free cDNA products, Mm RT- Cas1 / Cas2 was incubated with [ ^-32P]-dGTP in reaction medium containing 10 mM Mg2+and 1 mM Mn2+for 7.5 or 15 min and then chased the reactions with higher concentrations of unlabeled dGTP or an equimolar mix of all 4 dNTPs for 30 min. The products were then analyzed on a denaturing 20% polyacrylamide gel before or after digestion with Protease K. Phosphorimager scans of the gel showed that [ ^-32P]-dGTP covalently bound to RT-Cas1 Attorney Docket No.10046-551WO1 (Protease K-sensitive label in well) was chased into larger dissociated DNA products, near full-length cDNAs (marked by stars in gel lanes) by higher concentrations of all 4 dNTPs, and progressively longer dG oligomers extending up the gel lane by higher concentrations of dGTP in the absence but not presence of ddGTP (Fig.6D). The autoradiograms also showed a series of labeled bands that appeared after Protease K digestion of products from the initial labeling period, but were not visible in the dNTP-chase lanes, suggesting that they corresponded32P-dGTP or short32P-dG oligomers that remained covalently bound to RT- Cas1 at early time points, but were chased into larger cDNA products that dissociated from the protein at later time points (Fig.6D, see also fig.14B). Surprisingly, the autoradiogram also showed a series of intensely32P-labeled protease-insensitive lower molecular weight bands (up to ~20 nt) that were not appreciably chased into larger products by higher concentrations of dGTP or dNTPs. Further experiments showed the intensely labeled bands were non-protein-associated32P-dG oligomers as short as dinucleotides that accumulated over time in the absence but not appreciably in the presence of higher concentrations of dGTP or dNTPs at the beginning of the time course (fig. 14B). Instead, higher concentrations of dGTP at the beginning of the time course led to the synthesis of longer dG oligomers by reiterative copying of the CCC sequence, while higher concentrations of dNTPs at the beginning of the labeling period led to the synthesis of near full-length cDNAs of the RNA template (fig.14B). These findings suggest that short DNA oligomers synthesized de novo or by rapid release after protein priming at early time points could be used to prime synthesis of longer DNA products. Collectively, the above findings indicated that Mm RT-Cas1 / Cas2 can use protein- priming to initiate cDNA synthesis at 3’-proximal sites with different trinucleotide sequences, either reiteratively copying those sequences to generate DNA oligomers in the presence of a single complementary dNTP or synthesizing near full-length cDNAs initiated at the trinucleotide in the presence of all 4 dNTPs. Protein priming enabled initiation of cDNA synthesis with different efficiencies by any dNTP, including with dGTP at the 3’-proximal CCC containing a CC dinucleotide that is a preferred site for de novo initiation (41). These experiments also showed that Mm RT-Cas1 / Cas2 synthesizes non-protein-associated DNA oligomers, either de novo or by rapid release after protein priming, with short DNA oligomers synthesized at early time points potentially used as primers for synthesis of longer cDNA products. Although these results do not preclude use of a primase mechanism for initiation of cDNA synthesis at some sites, time courses comparing rates of cDNA synthesis from the 3’- blocked-R50CCC template with 500 ^M dNTPs in the absence or presence of 20 ^M dG2 Attorney Docket No.10046-551WO1 primer showed a lag for initiation of DNA synthesis in the absence of the dG2 primer, most likely reflecting the time needed to synthesize a dG oligomer primer before beginning processive cDNA synthesis (fig.14C). Mm RT-Cas1 / Cas2 can initiate cDNA synthesis at complementary 3’-proximal sites by using 2-nt DNA oligonucleotide primers To test systematically if Mm RT-Cas1 / Cas2 could use short DNA oligomer primers to initiate DNA synthesis at complementary sites in an RNA template, Mm WT RT- Cas1 / Cas2 was incubated with 3’-blocked RNA templates (R50NNN_ddC) with different 3’- proximal trinucleotide sequences (AAA, CCC, GGG, or UUU) and 5’-labeled dA2, dC2, dG2, or dT2dinucleotide primers. The reactions were done in the presence of high concentrations of the unlabeled dNTP matching the dinucleotide (e.g., dATP for the dA2primer) or an equimolar mixture of all 4 unlabeled dNTPs in the absence of Mn2+to minimize terminal transferase addition to the dN2primer (visible as short DNA ladders in some gel lanes; Fig. 7A). Analysis of the products in a denaturing 20% polyacrylamide gel showed that when incubated with high concentrations of the same unlabeled dNTP, RT-Cas1 / Cas2 reiteratively copied the complementary trinucleotide in each template, generating a ladder of DNA homopolymers extending up the gel lane for those RNA templates that contained the complementary trinucleotide but not for the other templates (Fig. 7A). When the reactions were done with an equimolar mixture of all 4 unlabeled dNTPs, RT-Cas1 / Cas2 switched from reiterative copying of the complementary 3’ trinucleotide to synthesis of prominent longer cDNAs extending up to ~50-nt cDNAs only for those templates that contained the complementary 3’ trinucleotide (including both R50AAA and R50GGG for the dT2 primer; Fig.7A). Sequencing of the cDNAs synthesized after the dNTP chase in parallel reactions with unlabeled dN2 primers confirmed that the initiation sites were complementary di- or trinucleotide sequences, with the complementary 3’-proximal trinucleotide sequence a major initiation site in all cases (Fig.8A). The dG2primer also showed a low background of cDNA start sites at U, A or G residues found by sequencing to reflect a background of de novo or protein-primed initiations rather than non-Watson-Crick pairings of the dG2 primer (Fig.8A) (53). The ability to use very short DNA primers to faithfully initiate cDNA synthesis at complementary sites is enabled by the strong strand-annealing activity of group II intron-like RTs (22). Attorney Docket No.10046-551WO1 Mm RT-Cas1 / Cas2 initiation sites in the absence of an added primer are dependent upon the stability of base-pairing interactions over several nucleotides To investigate factors that affect the choice of cDNA initiation sites in the absence of an added primer, Mm WT RT-Cas1 / Cas2 was incubated with same 3’-blocked R50NNN templates with different 3’-proximal trinucleotide sequences and an equimolar mix of 500 ^M [ ^-32P]-dCTP, dATP, dGTP, and dTTP in the absence or presence of Mn2+. Analysis of the products in a denaturing 20% polyacrylamide gel showed prominent labeled cDNAs of ~50 nt suggestive of initiation of cDNA synthesis at the 3’-proximal trinucleotide sequence for the 3’-blocked R50CCC template in the absence or presence of Mn2+, as well as for the mutant R50CGC and R50GGG templates in the presence of Mn2+(Fig.7B). By contrast, the most prominent labeled bands for the R50UUU and R50AAA templates in the absence or presence of Mn2+were a series of shorter cDNAs ranging in size from 30 to 40 nt, with little if any indication of initiation at the 3’-proximal UUU or AAA sequences (Fig.7B). To identify putative cDNA initiation sites, the cDNAs synthesized from the different RNA templates were sequenced in parallel reactions with unlabeled dNTPs in the presence or absence of Mn2+(Fig.8B). The sequencing confirmed that most (75-92%) of the cDNAs synthesized from the R50CCC_ddC and R50CGC_ddC templates in the presence or absence of Mn2+began at the 3’-proximal CCC and CGC trinucleotide sites, with <15% beginning opposite A or U residues (Fig.8B, rows 2 and 5). The first nucleotide of most of the cDNAs initiated on the CCC and CGC templates corresponded to a G residue, while the second nucleotide corresponded to a G residue for the CCC template and a C residue for the CGC template, as expected for de novo initiation (41) or protein priming at the 3’-proximal C residue of the trinucleotide in both templates (Fig.8B). None of the cDNAs synthesized from the CGC template began at the middle G residues, reflecting that dCTP is used less efficiently for de novo or protein-primed initiation of cDNA synthesis than dGTP (Fig.8B). Switching the 3’-proximal CCC to AAA or UUU resulted in cDNAs beginning at a nearby upstream AAUUCUU sequence, including but not limited to the 3’ UU sequence used for initiation of snapback DNA synthesis with an RNA template having a 3’ OH (Fig.5) and surprisingly few initiations within the 3’ AAA or UUU trinucleotide sequences (Fig.8B, rows 1 and 4). Initiation at these upstream sites agrees with the shorter sizes of32P-labeled cDNAs synthesized from these templates in denaturing polyacrylamide gels (Fig.7B). The favored nucleotide for initiating cDNA synthesis on these templates was dATP (59-70%) followed by dGTP, dTTP, and dCTP (Fig.8B). Attorney Docket No.10046-551WO1 Finally, switching the 3’-proximal trinucleotide to GGG also resulted in increased use of initiation sites within the nearby upstream AAUUCUU sequence but with a high proportion of cDNAs initiated at the 3’-proximal GGG sequence in the absence of Mn2+and with the 3’- proximal GGG becoming the predominant initiation site in the presence Mn2+(Fig.8B, row 3). In this case, the major, almost exclusively used initiating nucleotide in the presence of Mn2+was dCTP, which can form a stable base pair with G but is the least favored dNTP for protein priming and likely de novo initiation by RT-Cas1 / Cas2 (Fig.6B). Collectively, these findings indicate that in addition to proximity to the 3’ end of the RNA, the stability of base- pairing interactions over several nucleotides can override specific sequences for the choice of cDNA initiation sites on RNA templates, as expected for initiation of cDNA synthesis by annealing of short newly synthesized DNA oligomer primers. Mm RT-Cas1 / Cas2 initiates at 3’-proximal sites in the RNA segment of 3’-dA-tailed RNAs To investigate how cDNA synthesis is initiated on RNA protospacers with 3’-DNA tails, the effect of varying the lengths of the RNA and 3’-DNA-tail were tested in two different sets of 3’-blocked RNA protospacers, one set (R29-32CCC+dAn) corresponding to the 3’ end of the R50CCC RNA template used above to identify cDNA initiation sites (Fig. 5 and 8), and the other set (R29+dAn) corresponding to the R29 RNA used to analyze RNA protospacer integration with or without a 3’-dA tail (Fig.3). For both sets of templates, it was found that preferred initiation sites in the presence or absence of Mn2+were located toward the 3’ end of the RNA segment, with few initiations occurring within the 3’-dA tail (gels shown in Fig. 7C, sequencing results shown in Fig. 8C). The sequences showed that the major initiation sites for the R29-32CCC+dAn templates were clustered at or near the CCC trinucleotide, the favored initiation site for the R50CCC template, but with higher proportions of initiations outside the CCC sequence, while the major initiation sites for the R29+dAntemplates were at a 3’-proximal CC dinucleotide sequence with minor initiation sites elsewhere in the RNA segment and few or none in the 3’-dA tail (Fig.8C). The initiation of cDNA synthesis at 3’- proximal sites in the RNA segment upstream of the 3’-dA tail yields RNA / DNA duplexes with a single-stranded 3’-DNA overhang, which is favored for integration dsDNA protospacers into CRISPR arrays by Cas1 / Cas2 (54). Kinetic analysis of RNA and DNA protospacer integration shows preferred mechanisms Finally, to investigate the mechanism by which 3’-dN tailed RNA protospacers are integrated into CRISPR arrays, kinetic assays of spacer integration were carried out. For these Attorney Docket No.10046-551WO1 assays, a CRISPR hairpin DNA substrate was used in which the top and bottom strands were connected by a 5-nt linker, making it possible to identify products resulting from coupled cleavage-ligation of 5’-labeled protospacers into either or both strands by the length of labeled DNA fragments (Fig.9A). The reactions were done in the absence of Mn2+or dNTPs with 3 sets of R29-dAnor R30CCC-dAnoligonucleotide protospacers (denoted I, II, III) without or with a complementary DNA strand that leaves different length 3’-dA overhangs on both strands of duplex protospacers (Fig.9, B to D). For each set, the rates of integration by WT RT-Cas1 / Cas2 or RT∆-Cas1 / Cas2 were compared in parallel assays with 4 different 5’- labeled (denoted by *) protospacers: a 3’-dA-tailed RNA (*RNA-dAn), a single-strand *DNA, an *RNA-dAn / DNA duplex with the RNA strand labeled, and an identical RNA- dAn / *DNA duplex with the DNA strand labeled, making it possible to separately quantitate the integration of each strand (plots shown in Fig. 9, B to D). The integration of a stable *RNA-dA2 / cDNA duplex in a similar spacer integration assay was confirmed by gel electrophoresis of labeled integration products before and after RNase H digestion (fig.15). For Mm WT RT-Cas1 / Cas2 at a relatively low protospacer concentration (5 nM), the rates and amplitudes for appearance of the 5’-labeled 99-nt cleavage-ligation product resulting from spacer integration into both strands were with one exception higher for the *RNA-dAn / DNA or RNA-dAn / *DNA duplexes (closedcircles) than for their single-stranded *RNA-dAnor *ssDNA counterparts (open circles), as expected for duplexes enabling more rapid sequential ligation into the top- and bottom-strands (Fig.9B; time courses for all labeled cleavage products shown in fig. 16A). The amplitudes determined from the curve fits for integration of *RNA-dAnor the *RNA-dAn / DNA duplex (red open and closed circles, respectively) were lower than those for their *ssDNA or RNA-dAn / *DNA counterparts (open and closed circles, respectively; Fig. 9B), indicating less efficient integration of the RNA strand by WT RT-Cas1 / Cas2 under these conditions. When the same reactions were done with RT ^-Cas1 / Cas2, the rates for integration increased for all substrates tested (Fig.9C). However, the increases in rates and amplitudes were larger for the *RNA-dAn and *RNA-dAn / DNA duplexes (red open and closed circles, respectively) than for their *DNA counterparts (open and closed circles), resulting in more similar and in one case higher rates and amplitudes for the *RNA-dAnand *RNA-dAn / DNA protospacers than for their similarly configured *DNA counterparts (Fig.9C). These findings likely reflect that the RT domain impedes RNA-dN spacer integration when not directly coupled to cDNA synthesis and suggest there may be relatively little inherent difference in the efficiency of integration of dN-tailed RNA and DNA protospacers. Attorney Docket No.10046-551WO1 Notably, when the spacer integration reactions with WT RT-Cas1 / Cas2 were done at higher protospacer concentrations (250 nM), both the single-stranded *RNA-dAnand *ssDNA protospacers (open circles, respectively) had higher rates and amplitudes than did their labeled duplex counterparts (closed circles, respectively), with the higher efficiency for ssDNA protospacers particularly striking for 2 of the 3 protospacer configurations tested (Fig. 9D). These findings show that integration of single-strand dN-tailed RNA and ssDNA protospacers can be favored in vivo for abundant pathogen RNAs and ssDNA fragments generated by RecBCD or other phage defense nucleases (55–59). Parallel assays comparing the integration efficiencies of protospacers with an RNA-dAnstrand to all-DNA versions of the same protospacer show that in a number of cases differences in rates and amplitudes between each set of protospacers largely reflected the sequence of the protospacer rather than whether it was an RNA or DNA (fig.17 compare to Fig.9 and fig.16). Discussion Here, biochemical mechanisms underlying pathways that could be used by Mm RT- Cas1 / Cas2 for cDNA synthesis and site-specific integration of RNA protospacers into CRISPR arrays (Fig. 10) was elucidated. All pathways begin with RT-Cas1 / Cas2 using its terminal transferase activity to add short DNA tails to the 3’ ends of RNA fragment protospacers generated by RNases in vivo. In one set of pathways (Fig. 10, left), RT- Cas1 / Cas2 synthesizes near full-length cDNAs of 3’-dN-tailed RNAs by using multiple mechanisms to initiate cDNA synthesis at 3’-proximal sites of diverse RNAs, including de novo initiation, protein priming, and annealing of short exogenous or synthesized DNA oligomer primers. The resulting 3’-dN-tailed RNA / DNA duplexes with deoxynucleotides at the 3’ ends of both strands are then ligated into the CRISPR array by a mechanism analogous to that used by conventional Cas1 / Cas2 proteins to integrate dsDNA protospacers. In a second set of pathways favored at higher protospacer concentrations (Fig. 10, right), 3’-dN-tailed RNAs are integrated directly into the CRISPR array prior to cDNA synthesis. Reverse transcription of the 3’-dN-tailed RNA by RT-Cas1 / Cas2 could then occur after integration (left) or after linked integration / disintegration reactions at CRISPR insertion sites on opposite strands (right) with single-stranded gaps filled in by RT-Cas1 / Cas2 or host DNA Pol I (60). The latter mechanisms could also be used for the integration of suitably sized ssDNA protospacers generated by RecBCD or other phage defense nucleases (39, 57, 60). In all pathways, the resulting gapped DNA after protospacer integration is held together by RT- Cas1 / Cas2 possibly assisted by cellular DNA damage control proteins until their dissociation Attorney Docket No.10046-551WO1 by transcription-coupled DNA repair that fully integrates the newly acquired spacer into the CRISPR array (39). The analysis of these mechanisms explains known features of RNA- spacer acquisition by Mm RT-Cas1 / Cas2 in vivo and revealed two novel biochemical activities with potential biotechnological applications: the ability of an RT to use multiple mechanisms to synthesize near full-length cDNAs of diverse RNA templates without an added primer or fixed sequence requirements, and the ability of a DNA integrase to site- specifically integrate RNAs into DNA genomes by adding deoxynucleotides at crucial locations. The requirement for adding short 3’-dN tails to enable direct integration of RNA protospacers into a CRISPR array was suggested by the finding that spacers acquired by RT- Cas1 / Cas2 proteins in vivo have larger numbers of non-coded nucleotides at spacer-repeat junctions than do those acquired by Mm RT ^-Cas1 / Cas2 or conventional Cas1 / Cas2 proteins that acquire spacers from DNA (Fig.1B). Biochemical assays supported this mechanism by showing that the non-coded nucleotides at spacer-repeat junctions of spacers acquired by Mm RT-Cas1 / Cas2 in vivo matched the nucleotide preferences of Mm RT-Cas1 / Cas2 terminal transferase in vitro (Fig.1C and Fig.2) and that Mm RT-Cas1 / Cas2 could directly integrate single-stranded 3’-dN-tailed RNA protospacers into a CRISPR DNA both in coupled terminal transferase / spacer-ligation reactions in the presence of a single nucleotide (dATP) and in uncoupled spacer ligation reactions using synthetic RNA oligonucleotides with different length 3’-dA tails (Fig. 3). The latter assays showed that addition of a single 3’-dA residue enabled detectable RNA protospacer integration and addition of 2 to 6 3’-dA residues increased rates of RNA protospacer integration to levels comparable to those of ssDNA protospacers with the same nucleotide sequence (Fig.3). RNA protospacers with a 3’ OH needed for terminal transferase addition of DNA tails could be generated by cellular RNases, such as RNase III, which cleaves structured RNAs and directly generates RNA fragments with a 3’ OH (61); by phosphatase removal of 3’ phosphates left by other RNases; or by an inherent RT-Cas1 / Cas2 endonuclease activity that cleaves upstream of a 3’-blocked RNA, an activity reported for telomerase RT (62). Unlike Type I and II CRISPR systems, whose surveillance complexes, recognize target DNAs by binding a Protospacer Adjacent Motif (PAM) and then checking for complementarity to a seed region corresponding to the first ~5 nt of the guide RNA, the seed regions of Type III CRISPR systems surveillance complexes are internal and thus unlikely to be affected by non- coding nucleotides at the 5’- or 3’-end of the protospacer (63). This difference likely accounts Attorney Docket No.10046-551WO1 for why RTs are associated with Type III CRISPR systems but not CRISPR systems that utilize a PAM (64, 65). As desired for an enzyme whose biological function is to acquire RNA-derived spacers that defend against diverse pathogens, it was found that Mm RT-Cas1 / Cas2 could use multiple mechanisms to initiate cDNA synthesis on RNA protospacers at different 3’- proximal sites with no fixed sequence requirements. In addition to recently reported de novo initiation with a strong preference for initiating at CC sequences (41), it was found that Mm RT-Cas1 / Cas2 can use protein-priming to initiate at multiple 3’-proximal sites with any dNTP, greatly expanding the number of suitable cDNA initiation sites (Figs.6, 7 and 8). It was also found that Mm RT-Cas1 / Cas2, like other group II intron-related RTs (22), has a strong strand-annealing activity that enables it to initiate site-specific cDNA synthesis by using primers as short as dinucleotides (Fig. 7A). Short DNA oligomer primers could be generated in vivo by cellular enzymes, including RecBCD, which selectively degrades pathogen DNAs not only into ssDNA fragments long enough to be acquired as spacers, but also into ssDNA fragments as short as 3 to 6 nt that could be used to preferentially prime DNA synthesis on pathogen nucleic acids (59, 66). These findings show that Mm RT-Cas1 / Cas2 can also use de novo initiation or protein priming to synthesize its own short DNA oligomer primers that initiate DNA synthesis at complementary sequences on RNA or DNA templates (Figs. 6, 7 and 8). Supporting this mechanism, it was found that the stability of base-pairing interactions over several nucleotides plays a substantial role in the selection of cDNA initiation sites (Fig. 7 and 8). Most compelling was the finding that both CCC and GGG at the same 3’-proximal location in otherwise identical RNA templates were both preferred initiation sites for cDNA synthesis (Fig. 8B), even though dCTP is the least favored dNTP for protein priming (Fig. 6B) and likely also for de novo initiation. Initiation at G residues with dCTP at the 3’-proximal GGG sequence trumped initiation at sites with C, U, or A residues that were favored for cDNA initiation in otherwise identical templates that lacked a 3’ GGG (Fig.8B). Although the results do not exclude a primase-like mechanism, time courses for initiation of cDNA synthesis at a 3’-proximal CCC sequence with 500 ^M dNTPs showed a pronounced lag for initiation of cDNA synthesis compared to a parallel assay in the presence of 20 ^M dinucleotide dG2primer, which can reflect the time needed for RT-Cas1 / Cas2 to synthesize a short DNA primer (fig.14C). Unlike AbiK and Abi-P2 RTs, which synthesize random sequence DNA oligomers in the absence of an RNA or DNA template (20), the synthesis of short DNA oligomers by Mm Attorney Docket No.10046-551WO1 RT-Cas1 / Cas2 required the presence of an RNA template (Fig.4A and Fig. 6A). AbiK and Abi-P2 RTs have distinctive structural features that prevent binding of a template but leave the RT active site accessible to dNTPs (20) whereas group II intron RT apoenzymes have a tendency to fold into an inactive structure in which the RT active site is blocked until activated by template binding (67). The finding that group II intron, human LINE-1, telomerase, and retroviral RTs are capable of Mn2+-dependent de novo initiation of cDNA synthesis (41) shows that de novo synthesis of short DNA oligomer primers can be an inherent activity of many if not all RTs. The ability of RT-Cas1 / Cas2 to integrate RNA-dN / cDNA duplexes with overhanging single-stranded DNA tails into a CRISPR array is consistent with and in retrospect might have been predicted from the structures of dsDNA integration complexes of Cas1 / Cas2 from CRISPR systems that lack an associated RT (25, 34, 54). These structures showed the Cas1 / Cas2 proteins form a hexameric complex with two Cas1 homodimers on either side separated by a Cas2 homodimer. The latter forms a platform for binding a DNA duplex across the length of the complex via non-sequence specific base and phosphate-backbone interactions with splayed or overhanging single-stranded 3’-DNA ends of the two DNA strands inserted into Cas1 active sites on opposite sides of the complex. The findings that Mm RT-Cas1 / Cas2 initiates cDNA synthesis on dA-tailed RNAs at 3’-proximal sites within the RNA segment (Fig.8C) and adds small numbers of non-coded nucleotides to the 3’ ends of completed cDNAs (Fig.5) have the desirable effect of generating RNA / cDNA duplexes with single-strand DNA overhangs on both strands, potentially making them favorable substrates for integration by Cas1 / Cas2. The inability of Mm RT-Cas1 / Cas2 proteins to integrate RNA spacers without short DNA tails may reflects steric hindrance by 2’ OH groups that impede binding of 3’-terminal nucleotides at the Cas1 active sites, with this hindrance possibly less for the Thiomicrospira RT-Cas1 / Cas2, which was reported to inefficiently but detectably integrate RNAs lacking a 3’-dN-tail (14, 25). Previous findings showed that the Mm and Thiomicrospira RT-Cas1 / Cas2 differ from conventional Cas1 / Cas2 proteins in being able to efficiently integrate ssDNA as well as dsDNA protospacers into CRISPR arrays, likely reflecting structural differences in the RT- Cas1 / Cas2 integrase that relax strict requirements for protospacer binding (12, 25). Here these findings are extended by showing that Mm RT-Cas1 can efficiently integrate 3’-dN RNAs into CRISPR arrays, with integration of duplex protospacers favored at lower protospacer concentrations and single-stranded protospacers favored at higher protospacer concentrations. The favored integration of duplex protospacers at lower protospacer Attorney Docket No.10046-551WO1 concentrations can reflecte that they are more stably bound than single-stranded protospacers by phosphate-backbone contacts to both strands and / or rapid sequential binding of the two 3’-ends to Cas1 active sites on opposite sides of the complex. The steeper concentration dependence for integration of a single-stranded protospacer may reflect that its less constrained binding to one active site is rate-limiting at lower protospacers concentrations, but more readily overcome at higher protospacers concentrations. This concentration dependence may be a built-in mechanism that favors protospacer acquisition from abundant RNAs produced by invading pathogens as well as ssDNA fragments generated by degradation of invading pathogen DNAs by RecBCD or other phage defense nucleases (42, 57). Also noteworthy was that deletion of the RT domain in Mm RT∆-Cas1 / Cas2 increased the rate and amplitudes of protospacer integration for RNA-dN and RNA-dN / DNA-dN duplexes to a greater extent than similarly configured DNA-dN protospacers (Fig. 9 and fig. 16 and 17). This finding likely reflects that the RT domain impedes RNA spacer integration when not directly coupled to cDNA synthesis and suggests there may be little inherent difference in the integration efficiencies of dN-tailed RNA and DNA protospacers by the Cas1 / Cas2 integrase domain. Other possible mechanism for RNA protospacer acquisition include: (i) snap-back cDNA synthesis followed by RNase digestion of the single-strand RNA loop leaving an RNA / cDNA heteroduplex, and (ii) RNase H-digestion of the RNA strand of the initial RNA- dA / cDNA heteroduplex followed by second-strand DNA synthesis by RT-Cas1 or a host DNA polymerase to generate a dsDNA protospacer. However, snap-back DNA synthesis would not generate an RNA-DNA duplex with a DNA-tail at the 3’ end of the RNA strand, while RNase H digestion of the RNA strand of an initial RNA / cDNA or RNA-dN / cDNA duplex followed by second-strand DNA synthesis would result in dsDNA protospacers lacking the distinctive ^2 nt non-coded nucleotide tails added to and required for integration of RNA protospacers by Mm RT-Cas1 / Cas2 (Fig.1C and Fig.3). The spacers acquired by Mm WT RT-Cas1 / Cas2 in its native host were positively correlated with gene expression levels and integrated in either orientation into the CRISPR array with no specific sequence requirements or enrichment for specific regions of the RNA ((12) and fig. 11D). The finding that deletion of the RT domain abolished the correlation between spacer acquisition and gene expression levels (12) together with the characteristics of the integrated spacers suggest that spacer acquisition by WT RT-Cas1 / Cas2 is dependent primarily on RNA abundance, with little or no distinction between host and pathogen RNAs. The integration of protospacers in either orientation by Cas1 / Cas2 reflects that Type III Attorney Docket No.10046-551WO1 CRISPR systems have no requirement for a PAM, enabling double-stranded protospacers to bind the two Cas1 active sites in either orientation and for single-stranded protospacers to bind to either Cas1 active site for integration into the top or bottom strand. Ma et al. suggested that dsDNA protospacers with one 3’ end inserted into either strand of the CRISPR array could become fully integrated simply by coupled integration / disintegration reactions followed by transcription-coupled DNA repair (60). The finding that single-stranded 3’-dN RNAs can be site-specifically integrated into a CRISPR array raises the intriguing possibility that only the terminal transferase activity RT-Cas1 / Cas2 proteins might be required for RNA protospacer integration in bacteria containing an enzyme that could copy a short RNA sequence into DNA (e.g., another RT or a host DNA polymerase such as Pol I that has an innate ability to copy RNA (68); Fig.10, right). The acquisition of RNA-derived spacers in M. mediterranea required over-expression of RT-Cas1 / Cas2 and was not seen with endogenously expressed proteins (12). These findings may reflect that RT-Cas1 / Cas2 over- expression is activated as a last resort when pathogen RNAs reach high copy number by evading other cellular defense mechanisms. Under those circumstances, the over-expressed RT-Cas1 / Cas2 has no compunction about integrating host-derived RNA protospacers, potentially leading to autoimmunity and altruistic cell death. Phylogenetic analyses indicated that CRISPR-associated RTs evolved on at least three different occasions, with the largest and most ancient clade having an RT domain closely related to that of group II introns RTs, and the smaller more recently evolved clades having a RT-domain more closely related to those of Abi-P2 or retron RTs (69), both of which likely evolved earlier from a group II intron RT. RT-Cas1 fusion proteins comprise a single evolutionary-branch of CRISPR-associated RTs, while free-standing CRISPR-associated RTs in other branches are juxtaposed and transcribed in the same direction as genes encoding Cas6 and Cas1 / Cas2 proteins, suggesting that a functional link between these proteins preceded the fusions (13, 14). The phylogenetic branching patterns also suggested that fusion of RT to Cas1 occurred first, followed by more recent fusion to Cas6 and that both fusions have been horizontally transferred to different Type III and a few Type VI CRISPR systems, suggesting an inherent ability to function together with other Cas1 / Cas2 proteins (14, 65). Frequent horizontal transfer and rapid sequence divergence make it difficult to distinguish whether RT-Cas1 fusion occurred once or independently on multiple occasion, which would further support a selective advantage for coordinating linked functions (69). Mobile group II intron-encoded RTs, the likely ancestors of CRISPR-associated RTs, have throughout evolution exhibited remarkable flexibility to modulate their biochemical Attorney Docket No.10046-551WO1 activities and acquire additional domains that optimize their biological functions. Bacterial examples include diversity generating retroelement RTs, which preferentially mis- incorporate specific dNTPs into protein-coding regions to enable host-phage tropism switching (70, 71); retron RTs and abortive phage infection (abi) RTs, which use different mechanisms to synthesize ssDNAs thought to trigger phage defense mechanisms (18, 20); and group II intron-like 4 (G2L4) RTs, which evolved to function in double-strand break repair via microhomology-mediated end joining by optimizing the strong strand annealing activity of group II intron RTs (22). Further examples include human LINE-1 and other eukaryotic non-LTR retrotransposon RTs, which evolved variations of their reverse transcription and DNA integration mechanisms adapted to eukaryotic genomes and nuclear- cytoplasmic compartmentalization, and the core spliceosomal protein PRP8, which evolved from a group II intron-like RT and promotes RNA splicing by binding snRNAs derived from group II intron RNA domains (72). The flexibility and advantageous biochemical activities of group II intron-related RTs can be useful for biotechnological applications. In addition to transcriptional recording devices (23), group II intron and other non-LTR-retroelement RTs that use multiple cDNA initiation mechanisms analogous to those found here for RT-Cas1 are advantageous for genome engineering methods, such as prime editing (73–76). Such methods can benefit from using RTs that synthesize full-length cDNAs without an added primer, as well as from the high fidelity, processivity, and strand-displacement activity that are enabled by or engineered into the distinctive conserved structural features of these RTs (40). Further, the finding that the addition of short DNA segments enables Mm RT-Cas1 / Cas2 to site-specifically integrate RNA into a DNA genome suggests a general method for integration of RNA into genomes by other DNA integrases and recombinases. Materials and Methods Bioinformatic analysis of spacers acquired in vivo Spacer sequences were obtained from the SRA depository: Mm WT and RT ^- Cas1 / Cas2: SRP066108; Fs: SRR8102160, 163, 166-8, 170, 172-7, 194, 207; Vv: SRR8962131-134,137,138; Tt: SRR11818505-8, SRR12227011-4; Se: SRR16249482-3; St: SRR1595171-3, 176,187,198, 209. Raw reads were downloaded and merged if the dataset was comprised of pair-end reads by using fastq-join (v1.3.1, 12 / 15 / 2015). Spacers were identified as sequences between two repeats. Candidate spacers were mapped to the genome of the bacterial strain from which they were obtained (Mm MMB-1 NC_015276.1; Fs: E. coli Attorney Docket No.10046-551WO1 BL21 (DE3) NC_012947.1; Vv: E. coli HMS174 (DE3) NZ_LM993812.1; Tt: Tt phage phiFa / KO MH673671.2 and MH673672.2; Se: S. aureus RN4220 NZ_CP076105.1; St: St JIM8232 FR875178.1) by using Bowtie2 (v2.2.5, 03 / 09 / 2015) with the following parameters: --very-sensitive-local. After mapping, duplicate spacer sequences were removed by samtools (v1.12, 03 / 17 / 2021) using the parameter markdup -r. Spacers with unique sequences were then analyzed by using customized bash and R scripts to identify non-encoded nucleotides and nucleotide frequencies at different position. Density plots for spacer length distribution were plotted in R (v4.3.1, 06 / 16 / 2023). The numbers of spacers having non-coded A, C, G, and T residues at positions N1to N5from the assumed 3’ end of the encoded spacer sequence (Fig. 1C) and di-nucleotides frequencies at spacer-repeat junctions (fig. 11C) were calculated for acquired spacers with non-coded nucleotide at one end only. For fig. 11A, spacer sequences on opposite strands were rearranged so that all matched the sense-strand RNA sequence of the protein-coding or non-coding RNA gene to which they mapped, and the number of spacers with A, C, G, or T residues at the first 5 non-coded nucleotide positions was calculated and plotted as bar graphs in R. The distribution of spacer sequences across each gene was calculated as the percentile of the midpoint of each mapped spacer in the gene from which it originated (protein-coding or non-coding; host or viral; fig.11D). Spacers that mapped between annotated genes were not included in this analysis. Bacterial strains E. coli DH5 ^ (F–Φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rK–, mK+) phoA supE44 λ–thi-1 gyrA96 relA1) was used for cloning, and E. coli Rosetta2 (DE3) (F- ompT hsdSB(rB- mB-) gal dcm (DE3) pRARE2 (CamR)), Novagen) was used for protein expression. DNA and RNA constructs used for protein expression and spacer acquisition assays Mm WT RT-Cas1 and RT∆-Cas1 were expressed from plasmids pMalRF-RT-Cas1- cHis and pMalRF-RT∆-Cas1-cHis, respectively (12, 14). The expressed proteins have a maltose-binding protein (MBP) tag fused to their N-terminus via a non-cleavable rigid linker to increase their solubility and stability in the absence of bound nucleic acids (77) and a C- terminal 8x His tag. The amino acid at position 2 of Cas6-RT-Cas1 was changed from leucine to valine to accommodate the MBP fusion. Attorney Docket No.10046-551WO1 pET9Cas2, used to express Cas2 protein without an added tag, was constructed by PCR amplifying the Cas2 coding region using primers Cas2_pet_5 and Cas2_pet_3 from the plasmid pCassetteAv2_pBAD, which contains the M. mediterranea CRISPR03 array, RT- Cas1, and Cas2 cloned into the pBAD / Myc–His B backbone (Life Technologies) (12). The PCR product was then cloned between the NdeI and BamHI sites of pET9a (EMD Millipore). The cloned PCR-amplified DNA sequences were confirmed by Sanger sequencing. Plasmid CassetteAv2_pBAD (see above) was used to prepare an 88-bp CRISPR DNA substrate (40-bp leader, 35-bp repeat 1, and 13 bp of spacer 1) by PCR with primers MMB1Lead40-5 and MMB1crisp3-r1 using Phusion High-Fidelity DNA polymerase according to the suppliers’ protocol (New England Biolabs or Thermo Fisher). The substrate was labeled by PCR with 20 µCi [ ^-32P]-dCTP (3,000 Ci / mmol, Revvity), 120 µM dCTP, and 600 µM of the other three dNTPs and purified by electrophoresis in a 6% polyacrylamide / 8 M urea gel, cutting out the labeled band, and electro-eluting the DNA using midi D-Tube dialyzer cartridges (Novagen). The eluted DNA was concentrated by butanol extraction, purified by using an Zymo Oligo Clean and Concentrator kit, and quantitated against a [ ^-32P]-dCTP standard curve using a Beckman LS6500 liquid scintillation counter. A shorter 68-bp CRISPR DNA was prepared by annealing complementary oligonucleotides L20R1top and L20R1bot, which have biotin moieties at their 3’ ends. 10 µM of each oligonucleotide was mixed, heated to 82˚C for 2 min, and slowly cooled to room temperature. The 68-bp CRISPR DNA contains a 20-bp leader, 35-bp repeat, and 13 bp of spacer 1. A hairpin variant (HP-3) of the 68-nt CRISPR DNA was made by connecting the 3’ end of the top strand to the 5’ end of the bottom strand by a 5-nt linker (5’-TACAT) and omitting the 3’ biotin blocking group. The sequences of all oligonucleotides used in this study are shown in Table 1. Preparation of single-stranded and duplex protospacers used in spacer integration assays Synthetic oligonucleotide protospacers were 5’-labeled in 50-µl reactions containing 1 nmol oligonucleotide, 600 µCi [ ^-32P]-ATP (6,000 Ci / mmol; Revvity), 1 nmol ATP, 40 units T4 polynucleotide kinase (New England Biolabs) incubated for 45 min at 37 ^C in the manufacturer’s buffer and purified in a 10% polyacrylamide / 8 M urea gel. The gel area containing the oligonucleotide was cut out, crushed, and incubated in 1 ml of TE (10 mM tris-HCl (pH 7.5), 1 mM Ethylenediaminetetraacetic acid (EDTA)) per ~1.5 cm of gel slice at 4 ^C overnight on a rotator. The supernatant was removed, and the gel pieces were washed with an equal volume of TE. The combined supernatants were extracted repeatedly with Attorney Docket No.10046-551WO1 butanol until the volume was ≤200 µl. After clean up with a Zymo Oligo Clean and Concentrator kit, the labeled oligonucleotide was eluted in 40 to 60 µl distilled H2O and quantitated against a [ ^-32P]-ATP standard curve using a LS 6500 scintillation counter (Beckman-Coulter). Duplex protospacers consisted of synthetic 5’-labeled RNA or DNA oligonucleotides having complementary 29- or 30-nucleotide sequences with dA-tails that leave different length single-stranded 3’-dA overhangs on both strands. The oligonucleotides were annealed at 2.5 µM concentration each in 20 µl of 25 mM tris-HCl pH 7.5, 20 mM NaCl by heating to 94˚C for 30 s and slowly cooling (0.1˚C / s) to 25˚C followed by further incubation for 5 min at 25˚C and then placed on ice prior to use. Analysis of the annealed protospacers on a non- denaturing 15% polyacrylamide gel indicated that >95% were double-stranded. Protein expression and purification Protein expression plasmids were transformed into E. coli Rosetta2 (EMD Millipore) and plated on LB plates (Lysogeny Broth; 10 g tryptone, 5 g yeast extract, 10 g NaCl, 10 mM tris-HCl (pH 7.5), and 20 g agar (DIFCO) per liter dH2O) containing Ampicillin (Amp, 100 µg / ml) and Chloramphenicol (Cap, 25 µg / ml). Single transformed colonies were inoculated into 100-ml LB medium supplemented with the same concentrations of Amp and Cap in a 250-ml Erlenmeyer flask and incubated overnight with shaking at 37°C. For preparation of Mm RT-Cas1 proteins, six 4-liter flasks with one liter LB broth were each inoculated with 10 ml of the overnight culture and grown at 37°C with shaking to log phase (O.D.6000.6 to 0.8). IPTG (Isopropyl β-d-1-thiogalactopyranoside) was then added to a final concentration of 1 mM, and the cultures were incubated at 19°C for 20 to 24 h. Cells were harvested by centrifugation (Beckman-Coulter JLA-8.1 rotor, 20 min, 5,000 x g), and the pelleted cells were resuspended in 10 ml per g cells of A1 buffer (25 mM tris-HCl (pH 7.5), 500 mM NaCl, 10% glycerol, 10 mM ß-mercaptoethanol (BME) on ice. The cells were then incubated with lysozyme (1 mg / ml, 30 min, 4°C) and sonicated (Branson Sonifier 450; 3 bursts of 15 sec each with 15 sec between each burst) in an ice water bath. The lysate was cleared by centrifugation (Beckman-Coulter JA-14 rotor; 29,400 x g, 25 min, 4°C), and polyethyleneimine (PEI) was added to the supernatant with stirring on ice in six steps to a final concentration of 0.4%. After 10 min, precipitated nucleic acids were removed by centrifugation (JA-14 rotor; 29,400 x g, 25 min, 4°C), and proteins were precipitated from the supernatant by adding ammonium sulfate to 60% saturation and incubating on ice for 30 min. Proteins were collected by centrifugation (Beckman-Coulter JA-14 rotor; 29,400 x g, 25 Attorney Docket No.10046-551WO1 min, 4°C), dissolved in 20 ml A1 buffer, and filtered through a 0.45-µm polyethersulfone membrane (Whatman Puradisc). Protein purifications were done by using an ÄKTA start system (Cytiva). RT-Cas1 proteins were purified by loading the filtered crude protein onto an amylose column (5 ml; MBPTrap HP; Cytiva), washing with 50 ml of A1 buffer, followed sequentially by 30 ml of A1 plus 1.5 M NaCl and 30 ml of A1 buffer. Bound proteins were eluted with 50 ml of 10 mM maltose in A1 buffer. Fractions containing RT-Cas1 were identified by SDS-PAGE and pooled. The protein was then loaded onto a Nickel column (5 ml, HisTrap FF column; Cytiva) and eluted with a 100 ml 25 to 100 mM imidazole gradient. Peak fractions were identified by SDS-PAGE, pooled, and dialyzed into A1 buffer (10-kDa molecular weight cut off; SnakeSkin Dialysis Tubing; Thermo Fisher Scientific). The dialyzed protein was concentrated to >10 µM by using a 30K Pall concentrator and stored at -80˚C in small (25 to 50 µl) aliquots. The initial steps in the preparation of Cas2 expressed from pET9-Cas2 were similar to those for RT-Cas1, except that the cell paste was resuspended in H1 buffer (25 mM tris-HCl (pH 7.5), 100 mM KCl, 10% glycerol, 10 mM dithiothreitol (DTT)), and precipitated by adding ammonium sulfate to 40% saturation. The Cas2-containing supernatant after ammonium sulfate precipitation was loaded directly onto a heparin-Sepharose column (5 ml; HiTrap Heparin HP column; Cytiva) and eluted with a linear 100 mM to 1 M KCl gradient. Cas2 peak fractions (~800 mM KCl) were identified by SDS-PAGE and frozen in elution buffer for storage overnight at -80˚C. The peak fractions were thawed, diluted to ~125 mM KCl, and loaded onto a second 5-ml heparin-Sepharose column. Cas2 was eluted with a 100 to 400 mM KCl gradient, followed sequentially by extended washes with 400 mM KCl and a elution gradient from 400 mM to 1 M KCl to separate Cas2 from contaminating nucleases. Peak fractions were identified by SDS-PAGE, dialyzed against H1 buffer, and loaded onto a SP column (5 ml, HiTrap SP HP column; Cytiva) from which proteins were eluted with a 100 mM to 1 M KCl linear gradient. Peak fractions were identified by SDS-PAGE and stored at -80˚C. Protein concentrations were measured by using a Qubit Protein assay kit (Life Technologies) according to the manufacturer's protocol. Nucleic acid contamination was assessed by using Qubit ssDNA, dsDNA and ssRNA assay kits. ssDNA was below 1 ng / ml in purified protein preparations and RNAs and dsDNA were undetectable. All Proteins were >90% pure as assayed by SDS-PAGE. Attorney Docket No.10046-551WO1 Terminal transferase assays Purified RT-Cas1-8xHis protein (2 µM) was mixed with purified Cas2 (2 µM) in reaction medium containing 100 mM KCl, 100 mM NaCl, and 25 mM tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 1 mM BME, and 2% glycerol and the incubated at room temperature for 5 min before placing on ice prior to use. Assays were done in 20 µl of 25 mM tris-HCl (pH 7.5), 10 mM MgCl2, 5’-labeled DNA or RNA oligonucleotides (100 nM), and 1 mM of a single dNTP in equimolar MgCl2, and 1 mM MnCl2 added where indicated. Reactions were initiated by adding 5 µL of protein complex for a final concentration of 500 nM. For time courses, the reaction volume was scaled up 10-fold and 20 µL samples were taken at each time point (up to 2 h). Terminal transferase assays with unlabeled oligonucleotide substrates were done as above in 20 µl reaction medium containing 83 nM [ ^-32P]-dNTP (3,000 Ci / mmol, Revvity) and 250 µM of unlabeled dNTP in equimolar MgCl2. Reactions were incubated at 37°C for 1 h and stopped by adding phenol-chloroform-isoamyl alcohol (phenol- CIA, 25:24:1). The supernatant was mixed at a 2:1 ratio with loading dye (90% formamide, 20 mM EDTA, and 0.25 mg / ml bromophenol blue and xylene cyanole), and nucleic acids were analyzed on an 8% polyacrylamide / 8 M urea gel, which was dried and scanned with a phosphorimager (Typhoon, Cytiva). Molecular weight markers were a 5’-labeled 10-nt ssDNA ladder (Invitrogen) run in a parallel lane. Spacer integration assays Purified MBP-Cas6-RT-Cas1-8xHis (2 µM) protein was mixed with purified Cas2 (2 µM) in reaction medium containing 100 mM KCl, 100 mM NaCl, and 25 mM tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 1 mM BME, and 2% glycerol, incubated at room temperature for 5 min, and then placed on ice prior to use. Spacer integration reactions were done with RT-Cas1 / Cas2 complex (500 nM), CRISPR DNA (5 to 100 nM), and single-strand or duplex DNA or RNA oligonucleotides (5 or 250 nM) in 50 mM KCl, 50 mM NaCl, 20 mM tris-HCl, pH 7.5, 10 mM MgCl2and dNTPs (0.5 to 1 mM in equimolar MgCl2) and MnCl2 (1 mM) added as indicated for individual experiments. The reaction volumes for the time courses were either 250 µl when taking 20 µl samples at each time point, 120 µl when taking 10-µl samples, or 20 µl for individual samples. Up to eight time courses were run in parallel in a PCR apparatus. Reactions were initiated by adding the RT-Cas1 / Cas2 complex that had been preincubated with CRISPR DNA, incubating at 37°C for times up to 2 h, and stopping the reaction by adding phenol-CIA (experiments in Fig.2, A and B) or EDTA (25 mM) and 1.8-units Proteinase K, and incubating at 25˚C for 15 min (all other experiments). Attorney Docket No.10046-551WO1 The supernatant was mixed at a 2:1 ratio with loading dye (90% formamide, 20 mM EDTA, and 0.25 mg / ml bromophenol blue and xylene cyanole), and nucleic acids were analyzed on a 6% polyacrylamide / 8 M urea gel. Gels were dried, and scanned with a phosphorimager. Molecular weight markers were a 5’-labeled 10-nt ssDNA ladder (Invitrogen) run in a parallel lane. Time course gels for spacer acquisition assays using the hairpin CRISPR DNA were done at 30˚C and quantitated by exposing the autoradiogram for times at which none of the bands were saturated after scanning. Gels were analyzed in ImageQuant (Cytiva) by boxing labeled bands along with a background control above or below the labeled band. The fraction of product was determined from the counts in the labeled band minus the background relative to the total counts of all labeled products in the lane. The data for the largest band (154 nt), which is an intermediate that has a protospacer inserted into the top strand, was fit to the equation Y=[(a*k1) / (k2-k1)]*[exp(-k1*t)-exp(-k2*t)], where t=time and k1 and k2 are the rate constants for production and further reaction of the intermediate. The other two bands were fit to a single exponential equation. R2values for all curve fits are shown in Table 2. Reverse transcription assays Reactions were done with Mm WT RT-Cas1 / Cas2 protein (500 nM) and DNA or RNA template oligonucleotides (250 nM) in 10 or 20 µl of reaction medium (50 mM KCl, 50 mM NaCl, 25 mM tris-HCl (pH 7.5), and 10 mM MgCl2, with dNTPs added at 500 µM or 1 mM in equimolar MgCl2). For the reverse transcription assays in Fig. 7A, dinucleotide primers were 5’-labeled using T4 polynucleotide kinase (New England Biolabs) and added at 20 µM final concentration without pre-annealing to the template. cDNA synthesis assays with unlabeled primers or without primers were done with a dNTP mix containing 83 nM [ ^-32P]- dNTP (3,000 Ci / mmol, Revvity) and 20 µM of the same unlabeled nucleotide plus 500 µM of the other 3 dNTPs or 83 nM [ ^-32P]-dNTP (3,000 Ci / mmol) plus 500 µM of each dNTP, as specified for individual experiments. MnCl2was added where indicated at 1 mM final concentration. Reactions were incubated at 25˚C for 1 h and stopped by adding EDTA (25 mM) plus 1.8-units Protease K (New England Biolabs), and incubating at 25˚C for 15 or 30 min. The samples were mixed at a 2:1 ratio with loading dye (90% formamide, 20 mM EDTA, and 0.25 mg / ml bromophenol blue and xylene cyanole) and analyzed in a 20% polyacrylamide / 8 M urea gel, which was dried and scanned with a phosphorimager. Molecular weight markers were a 5’-labeled 10-nt ssDNA ladder (Invitrogen) run in a parallel Attorney Docket No.10046-551WO1 lane. Time courses were done as above for times up to 90 min while scaling up the sample volume 10-fold and taking 10 or 20 µl samples at each time point. In order to determine if RT-Cas1 / Cas2 produces a stable RNA / cDNA duplex, cDNA synthesis reactions in the absence of primer were done as described above using the R50CCC- ddC or D50CCC_ddC templates. The reactions were scaled up to 40 µl and stopped by adding EDTA (25 mM). The cDNA products were then purified by using a Zymo Oligo Clean and Concentrator kit and incubated for 20 min at 37˚C in the presence or absence of RNase H (5 units, New England Biolabs) or RNase A (2 µg, New England Biolabs) according to the manufacturer’s protocol followed by Protease K digestion as above. Samples were analyzed in a native 15% polyacrylamide gel, which was dried and analyzed with a phosphorimager. Protein-priming assays Purified RT-Cas1 protein (2 µM) was mixed with purified Cas2 (2 µM) in reaction medium containing 100 mM KCl, 100 mM NaCl, and 25 mM tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 1 mM BME, and 2% glycerol and incubated at room temperature for 5 min before placing on ice until ready for use. Protein-priming assays were done with 500 nM RT- Cas1 / Cas2 in the absence or presence of 250 nM DNA or RNA oligonucleotide templates in reaction medium containing 25 mM tris-HCl (pH 7.5) and 10 mM MgCl2, with 1 mM MnCl2 added where indicated. Reactions were initiated by adding 83 nM [ ^-32P]-dNTP (3,000 Ci / mmol; Revvity) in 25 mM tris-HCl (pH 7.5), 10 mM MgCl2and incubated at 25˚C for 15 min. Some reactions were chased by adding 500 µM of a single dNTP or all 4 dNTPs in 25 mM tris-HCl (pH 7.5), 10 mM MgCl2 buffer or 25 mM tris-HCl (pH 7.5), 10 mM MgCl2 buffer alone as a control followed by further incubation at 25˚C for 30 min. Some samples were treated with Protease K (1.8 units; New England Biolabs), Micrococcal Nuclease (MNase; 4 x105units in 1 x MNase buffer; New England Biolabs) or RNase A (2 µg, Monarch RNase A; New England Biolabs) and incubating for another 15 min at 25˚C. The reactions were stopped by adding 4x NuPAGE Sample Buffer (Invitrogen) or 4x Laemmli Buffer (Bio- Rad) to a final 1x concentration and heating to 90˚C for 3 min. Samples were analyzed on a 4-15% polyacrylamide NuPAGE Bis-tris gel (Thermo Fisher) in 1x morpholineethanesulfonic acid / SDS running buffer (Thermo Fisher) or on a 4-20% polyacrylamide Criterion gel (Bio-Rad) in 1x tris / glycine / SDS buffer (Bio-Rad) with Precision Plus Protein standards (Bio-Rad) as size markers. Protein gels were stained with Coomassie blue as described (78), dried, and scanned with a phosphorimager. Attorney Docket No.10046-551WO1 Coupled reverse transcription-spacer acquisition assays for RNase H digestion of duplex spacers cDNAs were synthesized by incubating 5’-labeled R29+dA2 or R29+dA2_ddC protospacers (250 nM) with RT-Cas1 / Cas2 (500 nM) and dNTPs (100 µM) in 40 µL of 50 mM KCl, 50 mM NaCl, 25 mM tris-HCl (pH 7.5), and 10 mM MgCl2in the presence or absence of MnCl2(30 µM) and a dinucleotide dG2or dT2primer (20 µM) for 1 h at 25˚C. Relatively low concentrations of MnCl2 and dNTPs were used to limit the RT-Cas1 terminal transferase activity. The samples were cleaned up with a Zymo Oligo Clean and Concentrator kit and eluted in 40 µl H2O. Spacer acquisition assays were done as above by incubating 5 µl of the purified protospacer with Mm RT-Cas1 / Cas2 (500 nM) and CRISPR hairpin DNA substrate (100 nM) for 1 h at 25˚C. After adding EDTA to 25 mM, the reaction products were treated with Protease K (0.32 units, 15 min, 37˚C) and cleaned up with a Zymo Oligo Clean and Concentration kit. The products were then eluted in 25 µl H2O and 10 µl were incubated with or without RNase H (12.5 units; New England Biolabs) according to the manufacturer’s protocol for 15 min at 37˚C. Samples were mixed at a 2:1 ratio with loading dye and analyzed on an 8% polyacrylamide / 8 M urea gel against a 5’-labeled New England Biolabs low molecular weight DNA ladder run in a parallel lane. Thermostable Group II Intron Reverse Transcriptase sequencing (TGIRT-seq) of cDNAs synthesized by Mm RT-Cas1 / Cas2 Mm RT-Cas1 / Cas2 reverse transcription reactions used to synthesize cDNAs for sequencing were done as above with 500 nM RT-Cas1 / Cas2, 250 nM RNA template and 500 µM dNTPs without or with 1 mM MnCl2 or 20 µM dinucleotide DNA primers in 160 µl of reaction medium (see above) for 1 h at 25˚C. The reaction was stopped by adding 20 µl of stop solution (20 mM tris-HCl (pH 7.5), 200 mM EDTA) with or without RNase A (0.8 µg / µl) as indicated and incubated for 15 min at 37˚C followed by digestion with Protease K (20 µl, 0.4 unit / µl) for 15 min at 37˚C. The products were cleaned-up with a Zymo Oligo Clean & Concentrator Kit. Nucleic acid concentrations were measured with a Qubit ssDNA Assay kit (Invitrogen) according to the manufacturer’s protocols, and 10-20 ng of cDNA product from each reaction was used for TGIRT-seq library preparation. For construction of TGIRT-seq libraries for identification of cDNA start sites, first- strand DNA synthesis was done by TGIRT template switching using a 34-nt RNA containing an Illumina R2 adapter sequence annealed to a complementary 35-nt R2 (R2R) DNA leaving a single nucleotide 3’ DNA overhang (an equimolar mixture of A, T, G and C) that promotes Attorney Docket No.10046-551WO1 template-switching by base pairing to the 3’ nucleotide of an acceptor nucleic acid, in this case a cDNA synthesized by RT-Cas1 / Cas2 (79, 80). The reaction was stopped by adding Protease K (1 unit; New England Biolabs) and 25 mM EDTA followed by clean-up with a Monarch PCR & DNA Cleanup Kit (New England Biolabs). Second-strand DNA synthesis was done by ligating 10 µM of a blunt-end duplex comprised of a 32-bp Illumina R1-3’SpC3 and 5’-phosphorylated R1R-3’SpC3 DNA (pre-annealed by incubating at 95˚C for 3 min and slowly cooling to 25˚C) using a Quick ligase kit (New England Biolabs) according to manufacturer’s protocol followed by clean-up as described above. The ligated dsDNA products were amplified by PCR using Phusion High-Fidelity PCR Master Mix with HF Buffer (New England Biolabs) with 200 nM of Illumina multiplex and index barcode primers (98˚C for 10 s pre-denaturation followed by 15 cycles of 98˚C for 5 s, 60˚C 10 s, 72˚C 15 s). The resulting cDNA libraries were cleaned up by using 1.4x AMPure XP beads (Beckman Coulter) and eluted in 25-µl H2O. One µl of the library was analyzed on a 2100 Bioanalyzer using a High Sensitivity DNA chip (Agilent) to assess product profiles and concentrations, and the remainder was sequenced on an Illumina Nextseq 550 to obtain ~1 million 2 x 75 nt paired-end reads per sample at the Genome Sequencing and Analysis Facility (GSAF) at the University of Texas at Austin. For read mapping, Illumina TruSeq adapters and PCR primer sequences were trimmed from the reads with Cutadapt (v3.5, 09 / 29 / 2021) sequencing quality score cut-off at 20; p- value <0.01) and reads <15 nt after trimming were discarded. Pair-ended reads were merged by using fastq-join. The merged reads were then mapped to the reverse complement of the corresponding template sequences using Bowtie2 with the parameters --very-sensitive-local -L 5. Mapped reads with 5 or fewer soft-clipped nucleotides were retrieved and re-aligned to the reverse complement of the template sequence using MAFFT (Multiple alignment using fast Fourier transform, v7.520, 03 / 22 / 2023,) with the following settings: --auto -- addfragments --reorder --keeplength --preservecase. Mapping of cDNA start sites onto the template sequence, stacked-bar graphs, and sequence logos of reads comprising ≥1% of all reads were made using R. For analysis of snap-back DNA synthesis products, reads containing the full-length template sequence linked to the 5’ end of a cDNA were collected and RNA template sequences were trimmed by using Cutadapt v3.5 with default settings. Trimmed reads were then mapped to the reverse complement of the corresponding template sequences using Bowtie2 with the parameters --very-sensitive-local -L 5. Mapped reads were retrieved and re- aligned to the reverse complement of the template sequence by using MAFFT with the Attorney Docket No.10046-551WO1 following settings: --auto --addfragments --reorder --keeplength --preservecase. Reads for snap-back DNA synthesis products comprising ≥5% of all mapped reads were plotted against the corresponding template sequences using Illustrator (Adobe). All sequencing data sets are listed in Table 3. Quantitation and statistical analysis Products of terminal transferase, spacer acquisition, and cDNA synthesis assays were quantitated using ImageQuant TL ver. 8.1 (Cytiva). Excel ver. 16 (Microsoft) was used to determine mean, median, and standard deviation values. Prism 10.0 (Graphpad Software) was used for curve fitting of quantitated assays to determine kobs, Amplitude, and R2values for the curve fits. R (v4.0.3) package ggplot2 was used to generate sequence logos from high- throughput sequencing data.

[0002] Attorney Docket No.10046-551WO1 TABLES Table 1. List of oligonucleotides used. RNA sequences are shown in bold type, DNA sequences in regular type. N, equimolar dATP, dTTP, dGTP, dCTP; 3ddC, 3’ dideoxy CTP; 3Phos, 3’ phosphate; 3INVdT, 3’ inverted dTTP; 5Phos, 5’ phosphate; 3SpC3, 3’ C3 Spacer. Attorney Docket No.10046-551WO1 Attorney Docket No.10046-551WO1 Attorney Docket No.10046-551WO1 Attorney Docket No.10046-551WO1 Attorney Docket No.10046-551WO1 Attorney Docket No.10046-551WO1 Attorney Docket No.10046-551WO1

[0003] Attorney Docket No.10046-551WO1 Table 2. List of R2values for curve fits. Data are for experiments shown in Figures 2, 3C, 9B-D, 12, 14C, 16A-C and 17A-C. Attorney Docket No.10046-551WO1 Attorney Docket No.10046-551WO1 Attorney Docket No.10046-551WO1 Table 3. Datasets. RNA template sequences are shown in bold type, DNA template sequences in regular type.3ddC, 3’ dideoxy CTP. TGIRT-seq dataset obtained in this study were deposited in the Sequence Read Archive (SRA) with the accession number PRJNA1003443. Attorney Docket No.10046-551WO1 Attorney Docket No.10046-551WO1 Attorney Docket No.10046-551WO1 Attorney Docket No.10046-551WO1 Attorney Docket No.10046-551WO1

[0004] 1O W155-64001.oNtekcoDyenrottA 569,A7N8 3J1,RRR436 5P S 15392 451247581939,41,077040861 241,28,-469A0N1 61, 5J8R1,7-42463990987807180R3 2 , 4 6 5 5 , 73,PRS617122,6,2,5,731,1118 R3 -872955419 5 4671R1951816,1,712,16 7 9,3,0 20S 2 8 8 7 5 1 1 1 2,1,13A 9 -N38 0J2RR09R730491,03 7 1 9 158,93 7 479,4 ,8023P S 1 8 1 4 2 4,501,3,7tc eejlp y y ,)los nlntyl dtnonrmPareoSec u'3 o osrfos no ee,'3io a qiendd dn sn n ec h( n r )h tBiBpn oSu(Na'de5ne' ea5'3ptipio ot t nSwlcni'5obn12≥ Attorney Docket No.10046-551WO1 REFERENCES 1. E. V. Koonin, K. S. Makarova, Evolutionary plasticity and functional versatility of CRISPR systems. Plos Biol.20, e3001481 (2022). 2. S. J. J. Brouns, M. M. Jore, M. Lundgren, E. R. Westra, R. J. H. Slijkhuis, A. P. L. Snijders, M. J. Dickman, K. S. Makarova, E. V. Koonin, J. van der Oost, Small CRISPR RNAs Guide Antiviral Defense in Prokaryotes. Science 321, 960–964 (2008). 3. M. P. Terns, R. M. Terns, CRISPR-based adaptive immune systems. Curr. Opin. 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Claims

Attorney Docket No.10046-551WO1 WHAT IS CLAIMED IS:

1. A method of integrating a target RNA into DNA, the method comprising modifying a target RNA by inserting or substituting one or more deoxynucleotide segments into the target RNA, then exposing the modified target RNA to a DNA integrase or DNA recombinase, wherein said DNA integrase or DNA recombinase integrates the target RNA into the DNA.

2. The method of claim 1, wherein the deoxynucleotide segments are 1 or more nucleotides in length.

3. The method of claim 2, wherein the deoxynucleotide segments comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more deoxynucleotides.

4. The method of claim 3, wherein the deoxynucleotides are contiguous.

5. The method of claim 4, wherein the deoxynucleotides are at a 3’ end of the target RNA.

6. The method of any one of claims 1-5, wherein the integrase is site-specific or non-site-specific.

7. The method of any one of claims 1-6, wherein the integrase comprises Cas1.

8. The method of claim 7, wherein the site-specific integrase comprises a Cas1 / Cas2 complex.

9. The method of any one of claims 1-8, wherein the integrase or recombinase is fused to a reverse transcriptase (RT).

10. The method of claim 9, wherein the RT is bacterial, eukaryotic, or viral in origin.

11. The method of claim 9, wherein the RT is a non-LTR-retroelement RT.

12. The method of claim 10, wherein the RT is not naturally occurring.

13. The method of claim 9, wherein the RT is associated with a CRISPR / Cas system.

14. The method of claim 9, wherein the RT is not associated with a CRISPR / Cas system.

15. The method of any one of claims 1-14, wherein the deoxynucleotide segment comprises at least one adenine deoxynucleotide.

16. The method of claim 15, wherein the deoxynucleotide segment comprises at least two adenine deoxynucleotides.Attorney Docket No.10046-551WO1 17. The method of any one of claims 1-16, wherein the target RNA comprises 10 or more deoxynucleotides.

18. The method of claim 17, wherein the target RNA comprises 20 or more deoxynucleotides.

19. The method of any one of claims 1-18, wherein the target RNA is integrated into the DNA without the use of an added primer.

20. The method of claim 1, wherein the deoxynucleotide segment is attached to the target RNA via a terminal transferase.

21. An isolated nucleic acid molecule comprising a genomic DNA with an integrated RNA target, wherein the RNA target comprises at least one or more deoxynucleotides that are not part of the genomic DNA.

22. The nucleic acid molecule of claim 21, wherein the deoxynucleotides which are not part of the genomic DNA are at a 3’ end of the RNA target.

23. The nucleic acid molecule of claim 21 or 22, wherein the deoxynucleotide(s) comprises at least one adenine deoxynucleotide residue.

24. The nucleic acid molecule of claim 21, wherein the integrated RNA target comprises at least one chemically modified deoxynucleotide.

25. The nucleic acid molecule of any one of claims 21-24, wherein the nucleic acid further comprises an integrase or recombinase.

26. The nucleic acid molecule of claim 25, wherein the integrase comprises Cas1.

27. The nucleic acid molecule of claim 26, wherein the integrase is fused to an RT.

28. The nucleic acid molecule of claim 26, wherein the integrase comprises an RT-Cas1 fusion.

29. The nucleic acid molecule of claim 26, wherein the integrase comprises a Cas1 / Cas2 complex.

30. A method of reverse transcribing all or part of a target RNA into a cDNA, the method comprising incubating the target RNA with a) an RT that can initiate cDNA synthesis by multiple mechanisms, wherein said multiple mechanisms comprise de novo initiation, protein primers, orAttorney Docket No.10046-551WO1 the use of short exogenous or RT-synthesized DNA oligonucleotide proteins; and b) dNTPs; wherein said incubation is carried out under conditions suitable for reverse transcription, thereby producing cDNA.

31. A method of reverse transcribing all or part of a target RNA into a cDNA, the method comprising incubating the target RNA with a) an RT that can initiate cDNA synthesis by multiple mechanisms, wherein said multiple mechanisms comprise de novo initiation, protein primers, or the use of short exogenous or RT-synthesized DNA oligonucleotide proteins; and b) dNTPs; wherein said incubation is carried out under conditions suitable for reverse transcription, thereby producing cDNA.

32. The method of claim 31, wherein the RT is part of an RT-Cas complex.

33. The method of claim 32, wherein said RT-Cas complex is an RT-Cas1 fusion protein.

34. The method of claim 33, wherein the RT-Cas1 fusion comprises Marinomonas mediterranea RT-Cas1 / Cas2.

35. The method of any one of claims 30-34, wherein incubating is done in presence of Mn2+.

36. The method of any one of claims 30-35, wherein cDNA synthesis is done without an added primer.

37. The method of any one of claims 30-36, wherein the method is carried out in vitro.

38. The method of any one of claims 30-36, wherein the method is carried out in a cell-free environment.

39. The method of any one of claims 30-36, wherein the method is carried out in a cell.

40. A method of determining RNA sequences, the method comprising the steps of: a) obtaining a sample comprising two or more different RNA sequences; b) reverse transcribing the RNA sequences using an RT that is capable of using multiple mechanisms for initiating reverse transcription at different 3' proximal sites that enable cDNA synthesis from diverse RNAs in proportion to their abundance; wherein said reverse transcribing is done under conditions suitable for reverse transcription, thereby producing cDNA;Attorney Docket No.10046-551WO1 c) adding adapters to cDNA produced in step b); and d) analyzing cDNA from step c) to determine RNA sequences.

41. The method of claim 40, wherein the method comprises RNA SEQ.

42. The method of claim 40 or 41, wherein cDNA is analyzed using next generation sequencing (NGS).

43. The method of any one of claims 40-42, wherein the RNA sample is a whole cell sample.

44. The method of any one of claims 40-42, wherein the RNA sample is a tissue sample.

45. The method of any one of claims 40-44, wherein said multiple mechanisms comprise de novo initiation, protein primers, or the use of short exogenous or RT-synthesized DNA oligonucleotide proteins.

46. The method of claim 45, wherein the exogenous or RT-synthesized DNA oligomer primer is 2, 3, 4, 5, or 6 nucleotides in length.

47. A method of editing target DNA using prime editing without an added primer, the method comprising: a) contacting the target DNA with a pegRNA complex and an RT that can initiate cDNA synthesis by a combination of mechanisms, including de novo initiation, protein priming, or the use of exogenous or RT-synthesized DNA oligomer primers; wherein said RT is capable of transcription without an added primer, and further wherein said pegRNA complex provides a replacement sequence for the target DNA; b) incubating the target DNA, pegRNA, and RT under conditions suitable for: i) binding of pegRNA complex to one strand of target DNA; ii) nicking an opposite strand of the target DNA from where the pegRNA complex is bound using a nickase, thereby forming a flap of target DNA, wherein said flap of target DNA comprises the sequence for editing; iii) allowing the replacement sequence of pegRNA to hybridize to the flap of target DNA;Attorney Docket No.10046-551WO1 iv) using RT to copy the remaining portion of the flap, thereby forming a double- stranded flap; v) removing the replacement sequence of the pegRNA so that edited target DNA hybridizes with the opposite strand of target DNA, wherein said opposite strand of target DNA now comprises a mismatch; and vi) repairing the mismatched opposite strand of target DNA so that both strands comprise edited target DNA.

48. The method of claim 47, wherein the RT is part of an RT-Cas complex.

49. The method of claim 48, wherein said RT-Cas complex is an RT-Cas1 fusion protein.

50. The method of any one of claims 47-49, wherein the nickase is a Cas molecule.

51. The method of claim 49, wherein the RT-Cas1 fusion comprises Marinomonas mediterranea RT-Cas1 / Cas2.

52. A composition comprising an isolated reverse transcriptase (RT), wherein said RT can initiate cDNA synthesis by multiple mechanisms, wherein said multiple mechanisms comprise de novo initiation, protein primers, or the use of short exogenous or RT-synthesized DNA oligonucleotide proteins to synthesize a full-length or near-full length cDNA copy of a target RNA.

53. The composition of claim 52, wherein the short exogenous or synthesized DNA primer is 2- 10 nucleotides in length.

54. The composition of claim 52 or 53, wherein the target RNA comprises one or more DNA segments.

55. The composition of claim 54, wherein the DNA segment is 1-6 nucleotides in length.

56. The composition of claim 54 or 55, wherein the DNA segment is on a 3’ end of the target RNA.

57. The composition of any one of claims 52-56, wherein the RT is part of an RT-Cas1 complex.

58. The composition of claim 52, wherein the near-full length cDNA is 80% or more the length of the target RNA.Attorney Docket No.10046-551WO1 59. The composition of claim 52, wherein said composition does not comprise an oligonucleotide primer that is more than 6 nucleotides in length.

60. The composition of any one of claims 52-59, wherein said composition comprises dNTPs.

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