Cr ispr / cas-related methods and compositions for treating herpes simplex virus

Abstract

Described herein are CRISPR / CAS-related systems, compositions, and methods for editing the RS1, RL2, and / or LAT genes in human cells, as well as cells and compositions, including cells edited according thereto.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201680074667.3, filed on October 28, 2016, entitled "CRISPR / CAS Related Methods and Compositions for Treating Herpes Simplex Virus". The original application was the national phase application of international application No. PCT / US2016 / 059502, which claimed priority to U.S. Provisional Patent Application No. 62 / 249,071 and U.S. Provisional Patent Application No. 62 / 249,159, filed on October 30, 2015.

[0002] Priority requirements

[0003] This application claims priority to U.S. Provisional Application No. 62 / 249,071, filed October 30, 2015, and U.S. Provisional Application No. 62 / 249,159, filed October 30, 2015, the contents of which are incorporated herein by reference in their entirety.

[0004] Government Interest Statement

[0005] This invention was made with funding from the National Institutes of Health under grant number 1R43A1120302-01. The government holds certain rights to this invention.

[0006] sequence list

[0007] This specification refers to the sequence list (a .txt file named "2016-10-28_084177_0133_ST25.txt" submitted electronically on October 28, 2016). The "2016-10-28_084177_0133_ST25.txt" file was generated on October 28, 2016, and has a file size of 11,189,396 bytes. The entire contents of the sequence list are incorporated herein by reference. Technical Field

[0008] This disclosure relates to CRISPR / CAS-related methods, compositions, and genome editing systems for editing target nucleic acid sequences (e.g., editing RS1, RL2, and / or LAT genes) and their application with herpes simplex virus (HSV). Background Technology

[0009] Herpes simplex virus (HSV), such as herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2), is a ubiquitous and highly contagious pathogen. HSV-1 typically causes intermittent, painful blistering in the mouth and mucous membranes. HSV-2 typically causes intermittent, painful blistering in the genital area. Herpes simplex virus can cause a lifelong, recurrent viral response.

[0010] Most people develop HSV-1 infection during childhood. In adulthood, up to 80% of the U.S. population is infected with HSV-1. The incidence of new HSV-1 infections is an average of 1.6 cases per 100 people per year (Langenberg et al., 1999; New England Journal of Medicine 341: 1432-1438). The most severe manifestations of HSV-1 infection include, for example, keratitis, encephalitis, and meningitis.

[0011] More than 500 million people worldwide are infected with HSV-2. Up to 23 million people are newly infected each year. In the United States, approximately one-fifth of adults are seropositive for HSV-2 infection (Xu et al., Abstract 42nd Annual Meeting of the Infectious Diseases Society of America 739; September 30, 2004). The prevalence of HSV-2 is increasing: since 1976, the presence of seropositive HSV-2 in U.S. adults has increased by 30% (Fleming et al., New England Journal of Medicine 1997; 337: 1105-11). HSV-2 infection increases the risk of HIV infection, especially in patients with active lesions.

[0012] Infection with HSV-1 and / or HSV-2 is permanent. Following initial infection with HSV-1 or HSV-2, the virus establishes a latent infection that persists throughout the host's life. Initial HSV-1 infection typically causes painful blistering of the oral mucosa (including the lips, mouth, and nose). Initial HSV-1 infection less commonly affects the anogenital region, causing painful blistering of the genital and anal mucosa. Initial HSV-2 infection typically causes painful blistering of the anogenital mucosa. Initial HSV-2 infection less commonly affects the oral cavity, causing painful blistering of the lips, mouth, and nose mucosa.

[0013] Following primary infection, latent infection is established by HSV-1 and HSV-2 in all subjects. Reactivation of HSV-1 or HSV-2 after the establishment of latent infection can occur at any point in a subject's life. Reactivation of HSV-1 or HSV-2 is more likely to occur in older adults and immunocompromised individuals, including those with cancer, those with HIV / AIDS, and those who have received solid organ or hematopoietic stem cell transplants.

[0014] Both HSV-1 and HSV-2 cause ocular herpes. Historically, HSV-1 has been the primary pathogen causing ocular herpes infections. However, in recent years, the global incidence of HSV-2-related ocular infections has been rising.

[0015] Ocular infections of HSV-1 or HSV-2 can affect the ocular epithelium, causing keratitis. Keratitis is the most common form of ocular herpes. HSV-associated keratitis is the most common cause of infectious blindness in developed countries (Dawson et al., Review of Ophthalmology, 1976; 21(2): 121-135). Worldwide, there are approximately 1.5 million cases of HSV-associated ophthalmopathy and 40,000 cases of HSV-associated blindness or severe monocular visual impairment each year (Krawczyk et al., PLOS ONE, 2015; 10(1): e0116800; Farooq and Shukla, 2012; Review of Ophthalmology, 57(5): 448-462). The incidence of ocular HSV infection appears to be rising in the United States (Liesegang et al., 1989; Archives of Ophthalmology, 107: 1155–1159; Baratz et al., 2009, Research in Ophthalmic Vision Science, 50e-abstract, 5044). There are 15.6 cases of epithelial keratitis per 100,000 people per year, and about 50,000 cases per year in the United States (Farooq and Shukla, 2012; Ophthalmology Review 57(5): 448-462).

[0016] Ocular herpes can also affect the retina, causing retinitis. HSV-associated retinitis has a lower incidence than HSV-associated keratitis, but carries a higher risk of permanent vision impairment. HSV-associated retinitis most commonly affects adults and can lead to acute retinal necrosis (ARN). ARN causes permanent vision impairment in more than 50% of subjects (Roy et al., Ocular Immunology and Inflammation, 2014; 22(3): 170-174).

[0017] Newborns are at high risk of severe HSV-1 and HSV-2 infections. These infections are transmitted from mother to fetus during delivery. The chance of mother-to-fetal transmission is highest when the mother has a primary HSV-1 or HSV-2 infection during pregnancy. The incidence of neonatal herpes is approximately 4–30 per 100,000 newborns (Brown ZA et al., 2003; JAMA; 289(2): 203–209; Dinh TH et al., 2008; Sexually Transmitted Diseases; 35(1): 19–21). Newborns may develop severe HSV-related keratitis, retinitis, encephalitis, and / or meningitis. Ocular herpes in newborns can lead to immediate, permanent vision loss. Ocular HSV exposes newborns to the risk of developing ARN later in life. There is no cure or preventative treatment for HSV-1 or HSV-2. Treatment is primarily administered during the acute phase of infection. Primary HSV-1 or HSV-2 infections can be treated with antiviral therapies, including acyclovir, valacyclovir, and famciclovir. These treatments can reduce viral shedding, reduce pain, and improve wound healing time. Reactivated latent infections may resolve without treatment (potentially self-limiting) or may be treated with antiviral therapies. Antiviral therapies may be given prophylactically in some cases, including during delivery for mothers with a recent infection or reactivated HSV-1 or HSV-2.

[0018] Vaccines for the prevention of HSV-1 and HSV-2 infections are being developed. However, the efficacy of vaccines has been limited in controlled clinical trials. Recent vaccines against HSV-1 and HSV-2 infections are only 35% effective in preventing HSV-1 infection (Belshe et al., 2012; New England Journal of Medicine 366(1):34-43).

[0019] Despite advances in antiretroviral therapy, treatment, prevention, and / or reduction of HSV-1 and HSV-2 infections remain necessary, particularly treatment, prevention, and / or reduction of HSV-1 and HSV-2-related ocular infections, including keratitis and retinitis. Therapies that cure, prevent, or treat HSV-1 and HSV-2 ocular infections would be superior to current standards of care. Summary of the Invention

[0020] The methods, genome editing systems, and compositions of this invention provide therapeutic, preventive, and / or mitigating measures for herpes simplex virus (HSV) infections, such as ocular infections.

[0021] The methods, genome editing systems, and compositions of this invention can be used to provide treatment, prevention, and / or relief of herpes simplex virus ocular infections (including ocular infections caused by herpes simplex virus type 1 (HSV-1) and / or herpes simplex virus type 2 (HSV-2) or their symptoms), or to treat, prevent, and / or relieve their symptoms, for example, by altering (e.g., knocking out and / or knocking down) one or more of the HSV-1 or HSV-2 viral genes, for example by knocking out and / or knocking down one, two, or three of the RS1, RL2, and / or LAT genes. RL2 includes an open chromatin region that is associated with the regulation of LAT gene and HSV-1 viral gene expression during the latent, reactivation, and lysis infection periods (J. Gen. Virol., January 2008; 89(Pt 1): 68-77).

[0022] The methods, genome editing systems, and compositions of this invention provide treatment, prevention, and / or relief for herpes simplex virus ocular infections or their symptoms by knocking out the RS1, RL2, and / or LAT genes, said herpes simplex virus ocular infections including ocular infections caused by HSV-1 and / or HSV-2. The methods, genome editing systems, and compositions of this invention provide treatment, prevention, and / or relief for herpes simplex virus ocular infections or their symptoms by knocking down the RS1, RL2, and / or LAT genes, said herpes simplex virus ocular infections including ocular infections caused by HSV-1 and / or HSV-2. The methods, genome editing systems, and compositions of this invention provide treatment, prevention, and / or relief for herpes simplex virus ocular infections or their symptoms by simultaneously knocking out and knocking down the RS1, RL2, and / or LAT genes, said herpes simplex virus ocular infections including ocular infections caused by HSV-1 and / or HSV-2.

[0023] The methods, genome editing systems, and compositions of this invention provide treatment, prevention, and / or mitigation of herpes simplex virus (HSV) ocular infections, including ocular infections caused by HSV-1 and / or HSV-2 or their symptoms, by altering one or more locations within the RS1, RL2, or LAT genes to cause them to be destroyed and / or eliminated from infected cells.

[0024] In one aspect, the methods, genome editing systems, and compositions of the present invention can be used to alter (e.g., knock out or knock down) the expression of one, two, or three of the RS1, RL2, and / or LAT genes by targeting genes (e.g., targeting non-coding or coding regions of said genes) to treat, prevent, and / or alleviate HSV-1 or HSV-2 infection.

[0025] In some embodiments, coding sequences (e.g., coding regions (also referred to as coding sequences in this invention)) of one, two, or three of the RS1, RL2, and / or LAT genes are targeted for alteration, knockout, and / or knockdown of expression. In some embodiments, the coding regions are early coding regions, such as early coding regions of the RS1, RL2, and / or LAT genes. For example, and not as a limitation, the methods, genome editing systems, and compositions of this invention are used to alter one, two, or three of the RS1, RL2, and / or LAT genes by targeting coding sequences (e.g., intron or exon sequences) of one, two, or three of the RS1, RL2, and / or LAT genes to treat, prevent, and / or alleviate HSV-1 or HSV-2 infection. In some embodiments, the genes (e.g., coding sequences of one, two, or three of the RS1, RL2, and / or LAT genes) are targeted to knock out and / or knock down one, two, or three of the RS1, RL2, and / or LAT genes, for example, to eliminate the expression of one, two, or three of the RS1, RL2, and / or LAT genes; and / or to knock out one or more copies of one, two, or three of the RS1, RL2, and / or LAT genes, for example, by introducing an alteration that includes a mutation (e.g., insertion or deletion) in one, two, or three of the RS1, RL2, and / or LAT genes. In some embodiments, the methods, genome editing systems, and compositions provide an alteration that includes an insertion or deletion in one, two, or three of the RS1, RL2, and / or LAT genes.

[0026] In some embodiments, early coding sequences of one, two, or three of the RS1, RL2, and / or LAT genes are targeted to knock out or knock down one, two, or three of the RS1, RL2, and / or LAT genes. In some embodiments, the targeting affects one or more copies of the RS1, RL2, and / or LAT genes. In some embodiments, the targeted knockout or targeted knockdown methods reduce or eliminate the expression of one, two, or more RS1, RL2, and / or LAT gene products. In some embodiments, the methods, genome editing systems, and compositions provide a change comprising an insertion or deletion in one, two, or three of the RS1, RL2, and / or LAT genes.

[0027] In another aspect, the methods, genome editing systems, and compositions include the RS1, RL2, and / or LAT genes, such as promoters, enhancers, introns, 5'UTRs, 3'UTRs, polyadenylation signals, and / or open chromatin regions. In some embodiments, the genes (non-coding sequences of RS1, RL2, and / or LAT) are targeted to knock out the genes, for example, to eliminate the expression of the genes, for example, to knock out one or more copies of the RS1, RL2, and / or LAT genes, for example, by introducing alterations, such as mutations (e.g., insertions or deletions) in the RS1, RL2, and / or LAT genes. In some embodiments, the methods, genome editing systems, and compositions provide alterations, such as insertions or deletions in the RS1, RL2, and / or LAT genes.

[0028] In some embodiments, altering (e.g., knocking out or knocking down) the RS1 gene means: (1) reducing or eliminating RS1 gene expression, (2) interfering with the activity and / or function of the transcriptional regulator ICP4 protein encoded by the RS1 gene, or (3) reducing or eliminating intracellular, serum, and / or brain parenchymal levels of the transcriptional regulator ICP4 protein.

[0029] In some embodiments, altering (e.g., knocking out or knocking down) the RL2 gene means: (1) reducing or eliminating RL2 gene expression, (2) interfering with the activity and / or function of the ICP0 protein encoded by the RL2 gene, and / or (3) reducing or eliminating intracellular, serum, and / or brain parenchymal levels of the ICP0 protein.

[0030] In some implementations, altering (e.g., knocking out or knocking down) the LAT gene means: (1) reducing or eliminating LAT gene expression, (2) interfering with the activity and / or function of the protein encoded by the LAT gene, and / or (3) reducing or eliminating the intracellular, serum, and / or brain parenchyma levels of the protein encoded by the LAT gene.

[0031] In some embodiments, the methods, genome editing systems, and compositions disclosed herein provide an alteration comprising disrupting the RS1, RL2, and / or LAT genes by inserting or deleting one or more nucleotides, the insertion or deletion of one or more nucleotides being mediated by a Cas9 molecule (e.g., an enzymatically active Cas9 (eaCas9), such as a Cas9 nuclease or Cas9 nickase) or a Cas9 fusion protein. This type of alteration is also referred to as “knockout” of the RS1, RL2, and / or LAT genes. In some embodiments, knockout of the RS1, RL2, and / or LAT genes comprises knocking out one or more copies of the RS1, RL2, and / or LAT genes, for example, by introducing an alteration comprising a mutation (e.g., insertion or deletion) in the RS1, RL2, and / or LAT genes. In some embodiments, the alteration comprises an insertion or deletion in the RS1, RL2, and / or LAT genes. In some embodiments, the targeted knockout method is mediated by non-homologous end joining (NHEJ), for example, using a CRISPR / Cas system comprising a Cas9 molecule (e.g., the eaCas9 molecule) or a Cas9 fusion protein. In some embodiments, the Cas9 molecule or Cas9 fusion protein is a Cas9 variant, for example, a Streptococcus pyogenes Cas9 variant or a Staphylococcus aureus Cas9 variant. In some embodiments, the Streptococcus pyogenes Cas9 variant is an EQR variant. In some embodiments, the Streptococcus pyogenes Cas9 variant is a VRER variant. In some embodiments, the targeted knockout method reduces or eliminates the expression of a functional RL2 gene product. In some embodiments, the targeted knockout method reduces or eliminates the expression of a functional LAT gene product. In some embodiments, the targeted knockout method reduces or eliminates the expression of a functional RS1 gene product.

[0032] In some embodiments, the methods, genome editing systems, and compositions disclosed herein provide alterations to the expression of the RS1, RL2, and / or LAT genes, wherein the alterations do not include nucleotide insertions or deletions in the RS1, RL2, and / or LAT genes. This type of alteration is also referred to as “knockdown” of the RS1, RL2, and / or LAT gene expression. In some embodiments, the method causes a reduction, decrease, suppression, or elimination of the expression of the RS1, RL2, and / or LAT genes. In some embodiments, the targeted knockdown method is mediated by a CRISPR / Cas system to alter (e.g., block, reduce, or decrease transcription) the transcription of the RS1, RL2, and / or LAT genes, wherein the CRISPR / Cas system comprises a Cas9 molecule (e.g., an enzymatically inactive Cas9 (eiCas9) molecule) or a Cas9-fusion protein (e.g., an eiCas9 fusion protein (e.g., an eiCas9 fused to a transcriptional repressor domain or a chromatin-modifying protein)). In some embodiments, non-coding regions of the RS1, RL2, and / or LAT genes (e.g., enhancer regions, promoter regions, 5'UTR, 3'UTR, polyadenylation signals, and / or open chromatin regions) are targeted to alter the expression of the RS1, RL2, and / or LAT genes. In some embodiments, open chromatin regions of the RL2 gene are targeted to alter the expression of the RL2 gene. In some embodiments, transcriptional regulatory regions (e.g., promoter regions (e.g., promoter regions controlling transcription of the RS1, RL2, and / or LAT genes)) are targeted to alter (e.g., knock down) the expression of the RS1, RL2, and / or LAT genes. In some embodiments, one or more gRNA molecules include a targeting domain configured to target an eiCas9 molecule or eiCas9 fusion protein sufficiently close to the transcriptional regulatory region (e.g., a promoter region (e.g., the promoter region controlling transcription of the RS1, RL2, and / or LAT genes)) to reduce, decrease, or inhibit the expression of the RS1, RL2, and / or LAT genes. In some embodiments, the coding region of the RL2 gene is targeted to alter (e.g., knock down) the expression of the RL2 gene. In some embodiments, the coding region of the RS1 gene is targeted to alter (e.g., knock down) the expression of the RS1 gene. In some embodiments, the coding region of the LAT gene is targeted to alter (e.g., knock down) the expression of the LAT gene. In some embodiments, the eiCas9 molecule is a Cas9 variant, such as a Streptococcus pyogenes Cas9 variant or a Staphylococcus aureus Cas9 variant. In some embodiments, the Streptococcus pyogenes Cas9 variant is an EQR variant. In some embodiments, the Streptococcus pyogenes Cas9 variant is a VRER variant. In some implementations, targeted knockdown methods reduce or eliminate the expression of functional RL2 gene products.In some implementations, targeted knockdown methods reduce or eliminate the expression of the functional LAT gene product. In some implementations, targeted knockdown methods reduce or eliminate the expression of the functional RS1 gene product.

[0033] In some embodiments, knocking down the RS1, RL2, and / or LAT genes can cure HSV infection. In some embodiments, knocking down the RS1, RL2, and / or LAT genes provides a functional cure for HSV infection. In some embodiments, knocking down the RS1, RL2, and / or LAT genes results in a sustained virological response to HSV infection.

[0034] In some embodiments, regions of the RS1, RL2, and / or LAT genes known to be integrated into the subject's genome are targeted for knockdown. In some embodiments, regions of the RS1, RL2, and / or LAT genes known not to be integrated into the subject's genome are targeted for knockout. In some embodiments, the method includes knocking out regions of the RS1, RL2, and / or LAT genes not integrated into the subject's genome.

[0035] Knockout, knockdown, and simultaneous knockout and knockdown of the RS1, RL2, and / or LAT genes can reduce HSV infection, replication, and packaging, and thus can treat, prevent, and / or reduce HSV infection. Knockout, knockdown, and simultaneous knockout and knockdown of the expression of the RS1, RL2, and / or LAT genes can, alone or in combination, cause any of the following: reduced HSV DNA production, reduced viral infectivity, reduced viral particle packaging, reduced viral shedding, and / or reduced production of viral proteins (e.g., ICP0 and / or ICP4 proteins) encoded by the RS1, RL2, and / or LAT genes. In some embodiments, the method includes simultaneously 1) knocking out and 2) knocking down two different regions of the RS1, RL2, and / or LAT genes, for example, 1) knocking down regions of the RS1, RL2, and / or LAT genes integrated into the subject's genome and 2) knocking out different regions of the RS1, RL2, and / or LAT genes not integrated into the subject's genome.

[0036] This invention discloses a genome editing system comprising a gRNA molecule and a Cas9 molecule. The gRNA molecule includes a targeting domain complementary to a target sequence of a herpes simplex virus (HSV) viral gene, wherein the target sequence of the HSV viral gene is selected from a group consisting of the RS1 gene, RL2 gene, and LAT gene. In some embodiments, the targeting domain is configured to form double-strand breaks or single-strand breaks at HSV target sites of approximately 500 bp, 450 bp, 400 bp, 350 bp, 300 bp, 250 bp, 200 bp, 150 bp, 100 bp, 50 bp, 25 bp, or 10 bp, thereby altering the HSV viral gene. In some embodiments, altering the HSV viral gene includes knocking out the HSV viral gene, knocking down the HSV viral gene, or simultaneously knocking out and knocking down the HSV viral gene.

[0037] In some embodiments, the targeting domain is configured to target the coding or non-coding regions of the HSV viral gene, wherein the non-coding regions include the promoter region, enhancer region, intron, 3'UTR, 5'UTR, or polyadenylation signal region of the HSV viral gene; and the coding regions include the early coding regions of the HSV viral gene.

[0038] In some embodiments, the targeting domain comprises a nucleotide sequence that is identical to or differs from nucleotide sequences selected from SEQ ID NOS: 208 to 58749 by no more than 3 nucleotides.

[0039] In some embodiments, the Cas9 molecule is a Streptococcus pyogenes Cas9 molecule, the genome editing system knocks out the HSV-1 RS1 gene, and the target domain includes a nucleotide sequence that is identical to or differs by no more than 3 nucleotides from a nucleotide sequence selected from the group consisting of SEQ ID NOS: 208 to 2509.

[0040] In some embodiments, the Cas9 molecule is a Streptococcus pyogenes Cas9 molecule, the genome editing system knocks out the HSV-2 RS1 gene, and the target domain includes a nucleotide sequence that is identical to or differs by no more than 3 nucleotides from a nucleotide sequence selected from the group consisting of SEQ ID NOS: 7098 to 9292.

[0041] In some embodiments, the Cas9 molecule is a Streptococcus pyogenes Cas9 molecule, the genome editing system knocks out the HSV-1 RL2 gene, and the target domain includes a nucleotide sequence that is identical to, or differs from, a nucleotide sequence selected from the group consisting of SEQ ID NOS: 21324 to 22744 by no more than 3 nucleotides.

[0042] In some embodiments, the Cas9 molecule is a Streptococcus pyogenes Cas9 molecule, the genome editing system knocks out the HSV-2 RL2 gene, and the target domain includes a nucleotide sequence that is identical to, or differs from, the nucleotide sequence selected from the group consisting of SEQ ID NOS: 26613 to 28037 by no more than 3 nucleotides.

[0043] In some embodiments, the Cas9 molecule is a Streptococcus pyogenes Cas9 molecule, the genome editing system knocks out the HSV-1LAT gene, and the target domain includes a nucleotide sequence that is identical to, or differs from, a nucleotide sequence selected from the group consisting of SEQ ID NOS: 31730 to 32746 by no more than 3 nucleotides.

[0044] In some embodiments, the Cas9 molecule is a Streptococcus pyogenes Cas9 molecule, the genome editing system knocks out the HSV-2LAT gene, and the target domain includes a nucleotide sequence that is identical to, or differs from, the nucleotide sequence selected from the group consisting of SEQ ID NOS: 35617 to 36926 by no more than 3 nucleotides.

[0045] In some embodiments, the Cas9 molecule is a Staphylococcus aureus Cas9 molecule, the genome editing system knocks out the HSV-1 RS1 gene, and the target domain includes a nucleotide sequence that is identical to or differs from nucleotide sequences selected from the group consisting of SEQ ID NOS: 2510 to 7073 by no more than 3 nucleotides.

[0046] In some embodiments, the Cas9 molecule is a Staphylococcus aureus Cas9 molecule, the genome editing system knocks out the HSV-2 RS1 gene, and the target domain includes a nucleotide sequence that is identical to or differs by no more than 3 nucleotides from a nucleotide sequence selected from the group consisting of SEQ ID NOS: 9293 to 13614.

[0047] In some embodiments, the Cas9 molecule is a Staphylococcus aureus Cas9 molecule, the genome editing system knocks out the HSV-1 RL2 gene, and the target domain includes a nucleotide sequence that is identical to, or differs from, the nucleotide sequence selected from the group consisting of SEQ ID NOS: 22745 to 26601 by no more than 3 nucleotides.

[0048] In some embodiments, the Cas9 molecule is a Staphylococcus aureus Cas9 molecule, the genome editing system knocks out the HSV-2 RL2 gene, and the target domain includes a nucleotide sequence that is identical to, or differs from, the nucleotide sequence selected from the group consisting of SEQ ID NOS: 28038 to 31720 by no more than 3 nucleotides.

[0049] In some embodiments, the Cas9 molecule is a Staphylococcus aureus Cas9 molecule, the genome editing system knocks out the HSV-1LAT gene, and the targeting domain includes a nucleotide sequence that is identical to, or differs from, a nucleotide sequence selected from the group consisting of SEQ ID NOS: 32747 to 35600 by no more than 3 nucleotides.

[0050] In some embodiments, the Cas9 molecule is a Staphylococcus aureus Cas9 molecule, the genome editing system knocks out the HSV-2LAT gene, and the targeting domain includes a nucleotide sequence that is identical to, or differs from, the nucleotide sequence selected from the group consisting of SEQ ID NOS: 36927 to 40871 by no more than 3 nucleotides.

[0051] In some embodiments, the Cas9 molecule is a Streptococcus pyogenes Cas9 molecule, the genome editing system knocks down the HSV-1 RS1 gene, and the target domain includes a nucleotide sequence that is identical to, or differs from, a nucleotide sequence selected from the group consisting of SEQ ID NOS: 13637 to 14794 by no more than 3 nucleotides.

[0052] In some embodiments, the Cas9 molecule is a Streptococcus pyogenes Cas9 molecule, the genome editing system knocks down the HSV-2 RS1 gene, and the target domain includes a nucleotide sequence that is identical to, or differs from, a nucleotide sequence selected from the group consisting of SEQ ID NOS: 17753 to 18784 by no more than 3 nucleotides.

[0053] In some embodiments, the Cas9 molecule is a Streptococcus pyogenes Cas9 molecule, the genome editing system knocks down the HSV-1 RL2 gene, and the targeting domain includes a nucleotide sequence that is identical to, or differs from, a nucleotide sequence selected from the group consisting of SEQ ID NOS: 40886 to 42078 by no more than 3 nucleotides.

[0054] In some embodiments, the Cas9 molecule is a Streptococcus pyogenes Cas9 molecule, the genome editing system knocks down the HSV-2 RL2 gene, and the targeting domain includes a nucleotide sequence that is identical to, or differs from, a nucleotide sequence selected from the group consisting of SEQ ID NOS: 49498 to 50652 by no more than 3 nucleotides.

[0055] In some embodiments, the Cas9 molecule is a Streptococcus pyogenes Cas9 molecule, the genome editing system knocks down the HSV-1LAT gene, and the targeting domain includes a nucleotide sequence that is identical to, or differs from, a nucleotide sequence selected from the group consisting of SEQ ID NOS: 45340 to 46479 by no more than 3 nucleotides.

[0056] In some embodiments, the Cas9 molecule is a Streptococcus pyogenes Cas9 molecule, the genome editing system knocks down the HSV-2LAT gene, and the targeting domain includes a nucleotide sequence that is identical to, or differs from, a nucleotide sequence selected from the group consisting of SEQ ID NOS: 53858 to 55056 by no more than 3 nucleotides.

[0057] In some embodiments, the Cas9 molecule is a Staphylococcus aureus Cas9 molecule, the genome editing system knocks down the HSV-1 RS1 gene, and the target domain includes a nucleotide sequence that is identical to, or differs from, a nucleotide sequence selected from the group consisting of SEQ ID NOS: 14795 to 17741 by no more than 3 nucleotides.

[0058] In some embodiments, the Cas9 molecule is a Staphylococcus aureus Cas9 molecule, the genome editing system knocks down the HSV-2 RS1 gene, and the target domain includes a nucleotide sequence that is identical to, or differs from, a nucleotide sequence selected from the group consisting of SEQ ID NOS: 18785 to 21311 by no more than 3 nucleotides.

[0059] In some embodiments, the Cas9 molecule is a Staphylococcus aureus Cas9 molecule, the genome editing system knocks down the HSV-1 RL2 gene, and the targeting domain includes a nucleotide sequence that is identical to, or differs from, a nucleotide sequence selected from the group consisting of SEQ ID NOS: 42079 to 45315 by no more than 3 nucleotides.

[0060] In some embodiments, the Cas9 molecule is a Staphylococcus aureus Cas9 molecule, the genome editing system knocks down the HSV-2 RL2 gene, and the target domain includes a nucleotide sequence that is identical to, or differs from, the nucleotide sequences selected from the group consisting of SEQ ID NOS: 50653 to 53824 by no more than 3 nucleotides.

[0061] In some embodiments, the Cas9 molecule is a Staphylococcus aureus Cas9 molecule, the genome editing system knocks down the HSV-1LAT gene, and the targeting domain includes a nucleotide sequence that is identical to, or differs from, a nucleotide sequence selected from the group consisting of SEQ ID NOS: 46480 to 49479 by no more than 3 nucleotides.

[0062] In some embodiments, the Cas9 molecule is a Staphylococcus aureus Cas9 molecule, the genome editing system knocks down the HSV-2LAT gene, and the targeting domain includes a nucleotide sequence that is identical to, or differs from, the nucleotide sequence selected from the group consisting of SEQ ID NOS: 55057 to 58731 by no more than 3 nucleotides.

[0063] In some embodiments, the Streptococcus pyogenes Cas9 molecule recognizes the protospacer adjacent motif (PAM) NGG.

[0064] In some embodiments, the Staphylococcus aureus Cas9 molecule recognizes either NNGRRT (SEQ ID NO:204) or NNGRRV (SEQ ID NO:205) PAM.

[0065] The present invention discloses a gRNA molecule, for example, an isolated or non-naturally occurring gRNA molecule, said gRNA molecule comprising a targeting domain complementary to a targeting domain (also referred to as a "target sequence") of the RS1, RL2, or LAT gene. The present invention discloses a composition comprising this gRNA molecule. Furthermore, the present invention discloses a vector comprising this gRNA molecule. Additionally, the present invention discloses cells comprising the genome editing system, vector, or composition disclosed herein. In some embodiments, the cells are selected from the group consisting of epithelial cells, nerve cells, and visual cells.

[0066] In some embodiments, the targeting domain of the gRNA molecule is configured to provide sufficient proximity to a cleavage event (e.g., a double-strand break or a single-strand break) of the HSV RS1, HSV RL2, or HSV LAT target to allow alterations to the HSV RS1, HSV RL2, or HSV LAT target (e.g., NHEJ-related alterations). In some embodiments, the targeting domain is configured such that the cleavage event (e.g., a double-strand break or a single-strand break) is located within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, or 200 nucleotides of the HSV RS1, HSV RL2, or HSV LAT target. The break (e.g., a double-strand or single-strand break) may be located upstream or downstream of the HSV RS1, HSV RL2, or HSV LAT target. In some embodiments, the targeting domain of the gRNA molecule is configured to provide a cleavage event selected from double-strand breaks and single-strand breaks within 500 (up to 500, 400, 300, 250, 200, 150, 100, 80, 60, 40, 20, or 10) nucleotides of the HSV RS1 target, HSV RL2 target, or HSV LAT target.

[0067] In some embodiments, the second gRNA molecule includes a second targeting domain configured to provide a cleavage event (e.g., a double-strand break or a single-strand break) sufficiently close to an HSV RS1 target, an HSV RL2 target, or an HSV LAT target to allow alterations to the HSV RS1 target, HSV RL2 target, or HSV LAT target (e.g., NHEJ-related alterations), either alone or in combination with a break localized by the first gRNA molecule. In some embodiments, the targeting domains of the first and second gRNA molecules are configured such that the cleavage event (e.g., a double-strand break or a single-strand break) for each gRNA molecule is independently located within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, or 200 nucleotides of the target. In some embodiments, the break (e.g., a double-strand or single-strand break) is located on either side of the HSV RS1 target, HSV RL2 target, or HSV LAT target. In some embodiments, the break (e.g., a double-strand or single-strand break) is located on one side of the HSV RS1 target, HSV RL2 target, or HSV LAT target, for example, upstream or downstream. In some embodiments, the targeting domain of the first and / or second gRNA molecule is configured to provide a cleavage event selected from double-strand and single-strand breaks within 500 nucleotides (within 500, 400, 300, 250, 200, 150, 100, 80, 60, 40, 20, or 10) of the HSV RS1 target, HSV RL2 target, or HSV LAT target.

[0068] In some embodiments, a single-strand break is accompanied by another single-strand break, which is localized by a second gRNA molecule, as described below. For example, the targeting domain is configured such that the cleavage event (e.g., two single-strand breaks) is located within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, or 200 nucleotides of the HSV RS1 target, HSV RL2 target, or HSV LAT target. In some embodiments, the first and second gRNA molecules are configured to be sufficiently close to each other that, upon guiding the Cas9 molecule or Cas9-fusion protein (e.g., Cas9 cleavage enzyme), the single-strand break is accompanied by another single-strand break (localized by a second gRNA molecule) to produce alterations to the HSV RS1, HSV RL2, or HSV LAT target. In some embodiments, the first and second gRNA molecules are configured such that, for example, when the Cas9 molecule or Cas9-fusion protein is a cleavage enzyme, a single-strand break located by the second gRNA is within 10, 20, 30, 40, or 50 nucleotides of the break located by the first gRNA molecule. In some embodiments, the two gRNA molecules are configured to cleave at the same position on different strands or within a few nucleotides of each other, essentially mimicking a double-strand break.

[0069] In some implementations, a double-strand break may be accompanied by another double-strand break, which is localized by a second gRNA molecule, as described below. For example, the targeting domain of the first gRNA molecule is configured such that the double-strand break is located upstream of the HSV RS1 target, HSV RL2 target, or HSV LAT target, for example, within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, or 200 nucleotides of the target, while the targeting domain of the second gRNA molecule is configured such that the double-strand break is located downstream of the HSV RS1 target, HSV RL2 target, or HSV LAT target, for example, within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, or 200 nucleotides of the target.

[0070] In some implementations, the double-strand break may be accompanied by two other single-strand breaks, which are localized by a second and a third gRNA molecule. For example, the targeting domain of the first gRNA molecule is configured such that the double-strand break is located upstream of the HSV RS1 target, HSV RL2 target, or HSV LAT target, for example, within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, or 200 nucleotides of the target, while the targeting domains of the second and third gRNA molecules are configured such that the two single-strand breaks are located downstream of the HSV RS1 target, HSV RL2 target, or HSV LAT target, for example, within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, or 200 nucleotides of the target. In some implementations, the targeting domains of the first, second, and third gRNA molecules are configured such that cleavage events (e.g., double-strand breaks or single-strand breaks) are independent for each of the gRNA molecules.

[0071] In some implementations, the first and second single-strand breaks may be accompanied by two other single-strand breaks, which are located by a third gRNA molecule and a fourth gRNA molecule. For example, the targeting domains of the first and second gRNA molecules are configured such that two single-strand breaks are located upstream of the HSV RS1 target, HSV RL2 target, or HSV LAT target, for example, within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, or 200 nucleotides of the target, while the targeting domains of the third and fourth gRNA molecules are configured such that two single-strand breaks are located downstream of the HSV RS1 target, HSV RL2 target, or HSV LAT target, for example, within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, or 200 nucleotides of the target. In some embodiments, the targeting domains of the first, second, third, and / or fourth gRNA molecules are configured to provide cleavage events selected from double-strand breaks and single-strand breaks within 500 (within 500, 400, 300, 250, 200, 150, 100, 80, 60, 40, 20, or 10) nucleotides of the HSV RS1 target, HSV RL2 target, or HSV LAT target.

[0072] In some implementations, multiple gRNAs are used to generate (1) two adjacent single-strand breaks, (2) two double-strand breaks, for example, bilaterally targeting HSV RS1, HSV RL2, or HSV LAT targets (e.g., to remove a segment of DNA, to create a deletion mutation), or to create more than one indel in the gene (e.g., in a coding region, an early coding region), (3) a double-strand break and two paired gaps, bilaterally targeting HSV RS1, HSV RL2, or HSV LAT targets (e.g., to remove a segment of DNA, to create an insertion deletion), or (4) four single-strand breaks (two on each side of the location), all targeting the same HSV RS1, HSV RL2, or HSV LAT target. In some implementations, multiple gRNAs can be used to target more than one HSV RS1, HSV RL2, or HSV LAT target.

[0073] In some embodiments, the targeting domains of the first gRNA molecule and the second gRNA molecule are complementary to the opposite strand of the target nucleic acid molecule. In some embodiments, the gRNA molecule and the second gRNA molecule are configured such that the PAM faces outward.

[0074] In some embodiments, the targeting domain of the gRNA molecule is configured to avoid unwanted target chromosomal elements, including but not limited to repetitive elements in the targeting domain, such as Alu repeats. As described in this invention, the gRNA molecule can be a first, second, third, and / or fourth gRNA molecule.

[0075] In some embodiments, the targeting domain of the gRNA molecule is configured to localize cleavage events sufficiently far from preselected nucleotides (e.g., coding nucleotides) to avoid altering the nucleotides. In some embodiments, the targeting domain of the gRNA molecule is configured to localize intron cleavage events sufficiently far from intron / exon boundaries or naturally occurring splicing signals to avoid altering exon sequences or unwanted splicing events. As described in this invention, the gRNA molecule may be a first, second, third, and / or fourth gRNA molecule.

[0076] In some embodiments, the targeting domain in the gRNA molecule targeting the HSV1 RS1 knockout site includes a nucleotide sequence that is identical to, or differs from, the nucleotide sequences selected from SEQ ID NO: 208 to 2509 and 2510 to 7073 by no more than one, two, three, four, or five nucleotides.

[0077] In some embodiments, the targeting domain in the gRNA molecule targeting the HSV2 RS1 knockout site includes a nucleotide sequence that is identical to, or differs from, the nucleotide sequences selected from SEQ ID NO:7098 to 9292 and 9293 to 13614 by no more than one, two, three, four, or five nucleotides.

[0078] In some embodiments, the targeting domain in the gRNA molecule targeting the HSV1 RL2 knockout site includes a nucleotide sequence that is identical to, or differs from, the nucleotide sequences selected from SEQ ID NO: 21324 to 22744 and 22745 to 26601 by no more than one, two, three, four, or five nucleotides.

[0079] In some embodiments, the targeting domain in the gRNA molecule targeting the HSV2 RL2 knockout site includes a nucleotide sequence that is identical to, or differs from, the nucleotide sequences selected from SEQ ID NO: 26613 to 28037 and 28038 to 31720 by no more than one, two, three, four, or five nucleotides.

[0080] In some embodiments, the targeting domain in the gRNA molecule targeting the HSV1 LAT knockout site includes a nucleotide sequence that is identical to, or differs from, the nucleotide sequences selected from SEQ ID NO: 31730 to 32746 and 32747 to 35600 by no more than one, two, three, four, or five nucleotides.

[0081] In some embodiments, the targeting domain in the gRNA molecule targeting the HSV2 LAT knockout site comprises a nucleotide sequence that is identical to, or differs from, the nucleotide sequences selected from SEQ ID NO:35617 to 36926 and 36927 to 40871 by no more than one, two, three, four, or five nucleotides.

[0082] In some embodiments, the targeting domain in the gRNA molecule targeting the HSV1 RS1 target knockdown site includes a nucleotide sequence that is identical to, or differs from, the nucleotide sequences selected from SEQ ID NO: 13637 to 14794 and 14795 to 17741 by no more than one, two, three, four, or five nucleotides.

[0083] In some embodiments, the targeting domain in the gRNA molecule targeting the HSV2 RS1 target knockdown position includes a nucleotide sequence that is identical to, or differs from, the nucleotide sequences selected from SEQ ID NO: 17753 to 18784 and 18785 to 21311 by no more than one, two, three, four, or five nucleotides.

[0084] In some embodiments, the targeting domain in the gRNA molecule targeting the HSV1 RL2 knockdown site includes a nucleotide sequence that is identical to, or differs from, the nucleotide sequences selected from SEQ ID NO:40886 to 42078 and 42079 to 45315 by no more than one, two, three, four, or five nucleotides.

[0085] In some embodiments, the targeting domain in the gRNA molecule targeting the HSV2 RL2 knockdown site includes a nucleotide sequence that is identical to, or differs from, the nucleotide sequences selected from SEQ ID NO:49498 to 50652 and 50653 to 53824 by no more than one, two, three, four, or five nucleotides.

[0086] In some embodiments, the targeting domain in the gRNA molecule targeting the HSV1 LAT knockdown site includes a nucleotide sequence that is identical to, or differs from, the nucleotide sequences selected from SEQ ID NO:45340 to 46479 and 46480 to 49479 by no more than one, two, three, four, or five nucleotides.

[0087] In some embodiments, the targeting domain in the gRNA molecule targeting the HSV2 LAT knockdown site includes a nucleotide sequence that is identical to, or differs from, the nucleotide sequences selected from SEQ ID NO: 53858 to 55056 and 55057 to 58731 by no more than one, two, three, four, or five nucleotides.

[0088] In some embodiments, the gRNA molecule is a single molecule or a chimeric gRNA molecule.

[0089] In some embodiments, the target domain of the gRNA molecule disclosed in this invention is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

[0090] In some embodiments, the gRNA molecule includes, from 5' to 3': a targeting domain (including a "core domain" and optionally a "secondary domain"); a first complementary domain; a linker domain; a second complementary domain; and a proximal domain. In some embodiments, the gRNA molecule also includes a tail domain. In some embodiments, the proximal domain and the tail domain together constitute a single domain.

[0091] In some embodiments, the gRNA molecule includes a linker domain of no more than 25 nucleotides in length, a proximal and tail domains of at least 20, 30, 35, or 40 nucleotides in length, and a targeting domain of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length.

[0092] Cleavage events (e.g., double-strand or single-strand breaks) can be generated by a Cas9 molecule or a Cas9-fusion protein. The Cas9 molecule or Cas9-fusion protein can be an enzymatically active Cas9 (eaCas9) molecule, such as an eaCas9 molecule that forms a double-strand break in the target nucleic acid or an eaCas9 molecule that forms a single-strand break in the target nucleic acid (e.g., a cleavage enzyme molecule). In some embodiments, the eaCas9 molecule can be a Cas9 variant. For example, but not as a limitation, the Cas9 variant can be a Streptococcus pyogenes Cas9 variant or a Staphylococcus aureus Cas9 variant. In some embodiments, the Streptococcus pyogenes Cas9 variant is an EQR variant. In some embodiments, the Streptococcus pyogenes Cas9 variant is a VRER variant. In addition to Cas9 molecules or Cas9-fusion proteins, other nucleases disclosed in this invention can also be used to generate cleavage events.

[0093] In some embodiments, the eaCas9 molecule or eaCas9-fusion protein catalyzes double-strand breaks.

[0094] In some embodiments, the eaCas9 molecule or eaCas9-fusion protein includes HNH-like domain cleavage activity but does not have or does not significantly have N-terminal RuvC-like domain cleavage activity. In this case, the eaCas9 molecule or eaCas9-fusion protein is an HNH-like domain cleavage enzyme, for example, the eaCas9 molecule or eaCas9-fusion protein includes a mutation at D10, such as D10A. In some embodiments, the eaCas9 molecule or eaCas9-fusion protein includes N-terminal RuvC-like domain cleavage activity but does not have or does not significantly have HNH-like domain cleavage activity. In some embodiments, the eaCas9 molecule or eaCas9-fusion protein is an N-terminal RuvC-like domain cleavage enzyme, for example, the eaCas9 molecule includes a mutation at H840, such as H840A. In some embodiments, the eaCas9 molecule or eaCas9-fusion protein is an N-terminal RuvC-like domain nickase, for example, the eaCas9 molecule or eaCas9-fusion protein includes a mutation at H863, such as H863A.

[0095] In some embodiments, single-strand breaks occur in the target nucleic acid in a strand complementary to the targeting domain of the gRNA molecule. In other embodiments, single-strand breaks occur in the target nucleic acid in a strand not complementary to the targeting domain of the gRNA molecule.

[0096] Furthermore, the subject matter disclosed in this invention provides a nucleic acid composition, such as an isolated or non-naturally occurring nucleic acid composition, such as DNA, said DNA comprising (a) a first nucleotide sequence encoding the gRNA molecule disclosed in this invention, such as a gRNA molecule comprising a targeting domain complementary to a target sequence of an RS1, RL2, or LAT gene, such as a target sequence at an HSV RS1 target, an HSV RL2 target, or an HSV LAT target.

[0097] In some embodiments, the nucleic acid composition further includes (b) a second nucleotide sequence encoding the Cas9 molecule or Cas9-fusion protein described in this invention. In some embodiments, the Cas9 molecule is an eiCas9 molecule. In some embodiments, the Cas9 molecule is an eaCas9 molecule. In some embodiments, the Cas9 molecule (e.g., an eiCas9 molecule or an eaCas9 molecule) may be a Cas9 variant, such as a Streptococcus pyogenes Cas9 variant or a Staphylococcus aureus Cas9 variant.

[0098] The Cas9 molecule or Cas9-fusion protein can be a nicking enzyme molecule, an enzyme-activated Cas9 (eaCas9) molecule, or an eaCas9-fusion protein, for example, an eaCas9 molecule or eaCas9-fusion protein that forms a double-strand break in the target nucleic acid and / or an eaCas9 molecule or eaCas9-fusion protein that forms a single-strand break in the target nucleic acid. In some embodiments, the single-strand break forms in the target nucleic acid in a strand complementary to the targeting domain of the gRNA molecule. In some embodiments, the single-strand break forms in the target nucleic acid in a strand that is not complementary to the targeting domain of the gRNA molecule.

[0099] In some embodiments, the nucleic acid composition further includes (c)(i) a third nucleotide sequence encoding the second gRNA molecule of the present invention, the third nucleotide sequence including a targeting domain complementary to a second target sequence of the RS1, RL2, or LAT gene; and optionally (c)(ii) a sequence encoding the third gRNA molecule of the present invention having a targeting domain complementary to a third target sequence of the RS1, RL2, or LAT gene; and optionally (c)(iii) a sequence encoding the fourth gRNA molecule of the present invention having a targeting domain complementary to a fourth target sequence of the RS1, RL2, or LAT gene.

[0100] In some embodiments, (a) and (b) are present on the same nucleic acid molecule, for example, on the same vector, for example, on the same viral vector, for example, on the same adeno-associated virus (AAV) vector or lentivirus (LV) vector. In some embodiments, the nucleic acid molecule is an LV vector. In some embodiments, the nucleic acid molecule is an AAV vector. Exemplary AAV vectors that can be used in any of the compositions and methods include AAV2 vectors, modified AAV2 vectors, AAV3 vectors, modified AAV3 vectors, AAV6 vectors, modified AAV6 vectors, AAV8 vectors, and AAV9 vectors. In some embodiments, the Cas9 molecule is an eiCas9 molecule. In some embodiments, the Cas9 molecule is an eaCas9 molecule. In some embodiments, the Cas9 molecule (e.g., an eiCas9 molecule or an eaCas9 molecule) may be a Cas9 variant, for example, a Streptococcus pyogenes Cas9 variant or a Staphylococcus aureus Cas9 variant.

[0101] In some embodiments, (a) it is present on a first nucleic acid molecule, for example, on a first vector, for example, on a first viral vector, for example, on a first AAV vector or a first LV vector; and (b) it is present on a second nucleic acid molecule, for example, on a second vector, for example, on a second vector, for example, on a second AAV vector or a second LV vector. The first and second nucleic acid molecules may be AAV vectors. In some embodiments, the first and second nucleic acid molecules may be LV vectors.

[0102] In some embodiments, (a) and (c)(i) are present on a nucleic acid molecule, for example, on a vector, for example, on a viral vector, for example, on an AAV vector or an LV vector. In some embodiments, the nucleic acid molecule is an AAV vector. In some embodiments, the nucleic acid molecule is an LV vector. In some embodiments, (a) and (c)(i) are on different vectors. In some embodiments, (a) is present on a first nucleic acid molecule, for example, on a first vector, for example, on a first viral vector, for example, on a first AAV vector or an LV vector; and (c)(i) is present on a second nucleic acid molecule, for example, on a second vector, for example, on a second AAV vector or a second LV vector. In some embodiments, the first and second nucleic acid molecules are AAV vectors. In some embodiments, the first and second nucleic acid molecules are LV vectors.

[0103] In some embodiments, each of (a), (b), and (c)(i) is present on a nucleic acid molecule, for example, on a vector, for example, on a viral vector, for example, on an AAV vector or an LV vector. In some embodiments, the nucleic acid molecule is an AAV vector. In some embodiments, the nucleic acid molecule is an LV vector. In some embodiments, one of (a), (b), and (c)(i) encodes on a first nucleic acid molecule, for example, on a first vector, for example, on a first viral vector, for example, on a first AAV vector or an LV vector; and the second and third of (a), (b), and (c)(i) encode on a second nucleic acid molecule, for example, on a second vector, for example, on a second AAV vector or an LV vector. The first and second nucleic acid molecules can be AAV vectors or LV vectors.

[0104] In some embodiments, (a) is present on a first nucleic acid molecule, for example, on a first vector, for example, on a first viral vector, for example, on a first AAV vector or LV vector; and (b) and (c)(i) are present on a second nucleic acid molecule, for example, on a second vector, for example, on a second AAV vector or LV vector. The first and second nucleic acid molecules may be AAV vectors or LV vectors.

[0105] In some embodiments, (b) is present on a first nucleic acid molecule, for example, on a first vector, for example, on a first viral vector, for example, on a first AAV vector or LV vector; and (a) and (c)(i) are present on a second nucleic acid molecule, for example, on a second vector, for example, on a second AAV vector or LV vector. The first and second nucleic acid molecules may be AAV vectors or LV vectors.

[0106] In some embodiments, (c)(i) is present on a first nucleic acid molecule, for example, on a first vector, for example, on a first viral vector, for example, on a first AAV vector or LV vector; and (b) and (a) are present on a second nucleic acid molecule, for example, on a second vector, for example, on a second vector, for example, on a second AAV vector or LV vector. The first and second nucleic acid molecules may be AAV vectors. In some embodiments, the first and second nucleic acid molecules may be LV vectors.

[0107] In some embodiments, each of (a), (b), and (c)(i) is present on a different nucleic acid molecule, for example, on a different vector, such as a different viral vector, or a different AAV vector or LV vector. For example, (a) may be on a first nucleic acid molecule, (b) on a second nucleic acid molecule, and (c)(i) on a third nucleic acid molecule. The first, second, and third nucleic acid molecules may be AAV vectors. In some embodiments, the first, second, and third nucleic acid molecules may be LV vectors.

[0108] In some embodiments, when a third and / or fourth gRNA molecule is present, each of (a), (b), (c)(i), (c)(ii), and (c)(iii) may be present on a nucleic acid molecule, for example, on a vector, for example, on a viral vector, for example, on an AAV vector or an LV vector. In some embodiments, the nucleic acid molecule is an AAV vector. In some embodiments, the nucleic acid molecule is an LV vector. In some embodiments, each of (a), (b), (c)(i), (c)(ii), and (c)(iii) may be present on different nucleic acid molecules, for example, on different vectors, for example, on different viral vectors, for example, on different AAV vectors or different LV vectors. In other embodiments, each of (a), (b), (c)(i), (c)(ii), and (c)(iii) may be present on more than one but fewer than five nucleic acid molecules, for example, on AAV vectors or LV vectors.

[0109] In some embodiments, the second gRNA molecule is configured to provide a cleavage event (e.g., a double-strand break or a single-strand break) sufficiently close to the HSV RS1 target, HSV RL2 target, or HSV LAT target to cause, alone or in combination with a break localized by the first gRNA molecule, alteration of the HSV RS1 target, HSV RL2 target, or HSV LAT target (e.g., NHEJ-related alteration).

[0110] In some embodiments, the third gRNA molecule is configured to provide a cleavage event (e.g., a double-strand break or a single-strand break) sufficiently close to the HSV RS1 target, HSV RL2 target, or HSV LAT target to allow alterations to the HSV RS1 target, HSV RL2 target, or HSV LAT target (e.g., NHEJ-related alterations) alone or in combination with breaks localized by the first and / or second gRNA molecules.

[0111] In some embodiments, the fourth gRNA molecule is configured to provide a cleavage event (e.g., a double-strand break or a single-strand break) sufficiently close to the HSV RS1 target, HSV RL2 target, or HSV LAT target to allow alterations to the HSV RS1 target, HSV RL2 target, or HSV LAT target (e.g., NHEJ-related alterations) alone or in combination with breaks localized by the first gRNA molecule, the second gRNA molecule, and / or the third gRNA molecule.

[0112] In some embodiments, the second gRNA is selected to target the same HSV RS1, HSV RL2, or HSV LAT targets as the first gRNA molecule. In some embodiments, the third and fourth gRNA molecules are selected to target the same HSV RS1, HSV RL2, or HSV LAT targets as the first and second gRNA molecules.

[0113] The targeting domains of the second, third, and fourth gRNA molecules may be individually selected from the nucleotide sequences shown in SEQ ID NO: 208 to 58749. The second, third, and fourth gRNA molecules may be modular gRNA molecules or chimeric gRNA molecules.

[0114] One or more nucleic acids and / or nucleic acid compositions present in the nucleic acid compositions of the present invention may include a promoter operatively linked to the nucleotide sequence, the nucleotide sequence encoding the gRNA molecule of (a), for example, the promoter of the present invention. The nucleic acid and / or nucleic acid compositions may also include a second promoter operatively linked to the nucleotide sequence, the nucleotide sequence encoding the second, third, and / or fourth gRNA of (c), for example, the promoter of the present invention. The promoter and the second promoter are different from each other. In some embodiments, the promoter and the second promoter are the same.

[0115] The nucleic acid composition of the present invention may further include a promoter operatively linked to the nucleotide sequence, the nucleotide sequence encoding the Cas9 molecule or Cas9-fusion protein of (b), for example, the promoter of the present invention.

[0116] The subject matter disclosed in this invention also provides a composition comprising (a) a gRNA molecule disclosed herein, for example, a gRNA molecule comprising a targeting domain complementary to a target sequence of the RS1, RL2, or LAT gene. In some embodiments, the composition further comprises (b) a Cas9 molecule (e.g., an eaCas9 molecule or an eiCas9 molecule) or a Cas9-fusion protein (as described herein). In some embodiments, the Cas9 molecule (e.g., an eaCas9 molecule or an eiCas9 molecule) may be a Cas9 variant. For example, but not as a limitation, the Cas9 variant may be a Streptococcus pyogenes Cas9 variant or a Staphylococcus aureus Cas9 variant. In some embodiments, the Streptococcus pyogenes Cas9 variant is an EQR variant. In some embodiments, the Streptococcus pyogenes Cas9 variant is a VRER variant. In some embodiments, the composition further comprises (c) a second, third, and / or fourth gRNA molecule, for example, the second, third, and / or fourth gRNA molecule described herein. In some embodiments, the composition is a pharmaceutical composition. For example, according to the methods disclosed in this invention, the compositions of this invention (e.g., the pharmaceutical compositions of this invention) can be used to treat, prevent, and / or alleviate HSV-1 or HSV-2 infection in subjects.

[0117] The subject matter disclosed in this invention also provides a method for altering HSV viral genes, wherein the HSV viral genes are selected from the group consisting of RS1, RL2, and LAT genes in cells, the method comprising administering one of the following groups to the cells:

[0118] (i) A gene editing system comprising a gRNA molecule and at least one Cas9 molecule, the gRNA molecule comprising a targeting domain complementary to a target sequence of the HSV viral gene;

[0119] (ii) a vector comprising a polynucleotide encoding a gRNA molecule and a polynucleotide encoding a Cas9 molecule, wherein the gRNA molecule includes a targeting domain complementary to a target sequence of the HSV viral gene; or

[0120] (iii) A composition comprising a gRNA molecule and at least one Cas9 molecule, the gRNA molecule comprising a targeting domain complementary to a target sequence of the HSV viral gene.

[0121] In another aspect, the present invention discloses a method for altering RS1, RL2, or LAT genes in cells, for example, by altering the structure of the target nucleic acid of the cell, for example, by altering the sequence of the target nucleic acid of the cell, said method comprising contacting said cells with: (a) a gRNA molecule disclosed in the present invention; and (b) a Cas9 molecule (e.g., eaCas9 molecule) or a Cas9-fusion protein, for example, the Cas9 molecule described in the present invention; and optionally, (c) a second, third, and / or fourth gRNA molecule targeting RS1, RL2, and LAT genes, for example, the second, third, and / or fourth gRNA molecule described in the present invention. In some embodiments, said Cas9 molecule may be a Cas9 variant.

[0122] In some embodiments, the method includes contacting cells from a subject who has or may develop HSV-1 and / or HSV-2. The cells may be from a subject who benefits from having a mutation at the HSV RS1 target, HSV RL2 target, or HSV LAT target.

[0123] In some implementations, the contact step can be performed in vivo.

[0124] In some embodiments, the method for altering cells according to the present invention includes, prior to the contact step, obtaining the sequences of HSV RS1 targets, HSV RL2 targets, or HSV LAT targets in the cells. Obtaining the sequences of the HSV RS1 targets, HSV RL2 targets, or HSV LAT targets in the cells can be achieved by sequencing one or more of the RS1, RL2, or LAT genes, or a portion of the RS1, RL2, or LAT genes.

[0125] In some embodiments, the contacting step of the method includes contacting the cells with a nucleic acid composition (e.g., a vector expressing at least one of (a), (b), and (c), such as an AAV vector or an LV vector). In some embodiments, the contacting step of the method includes contacting the cells with a nucleic acid composition (e.g., a vector expressing each of (a), (b), and (c), such as an AAV vector or an LV vector). In some embodiments, the contacting step of the method includes delivering to the cells a Cas9 molecule or a Cas9-fusion protein of (b), and a nucleic acid composition encoding a gRNA molecule of (a), and optionally, a second gRNA molecule encoding (c)(i), and further optionally, a third gRNA molecule encoding (c)(ii) and / or a fourth gRNA molecule encoding (c)(iii).

[0126] In some embodiments, the contacting step includes contacting the cells with a nucleic acid composition (e.g., an AAV vector or an LV vector) expressing at least one of (a), (b), (c), and (d). In some embodiments, the contacting step of the method includes contacting the cells with a nucleic acid composition (e.g., a vector or an AAV vector) expressing each of (a), (b), and (c). In some embodiments, the contacting step of the method includes delivering to the cells a Cas9 molecule or Cas9-fusion protein of (b) and a nucleic acid composition encoding a gRNA molecule of (a) and a template nucleic acid of (d), and optionally, a second gRNA molecule encoding (c)(i), and further optionally, a third gRNA molecule encoding (c)(iv) and / or a fourth gRNA molecule encoding (c)(iii).

[0127] In some embodiments, the contacting step includes contacting the cells with a nucleic acid composition, the nucleic acid composition being, for example, a vector, such as an AAV vector, such as an AAV2 vector, a modified AAV2 vector, an AAV3 vector, a modified AAV3 vector, an AAV6 vector, a modified AAV6 vector, an AAV8 vector, or an AAV9 vector. In some embodiments, the vector is an LV vector.

[0128] In some embodiments, contact includes delivering to the cells a Cas9 molecule or Cas9-fusion protein as a protein or mRNA (b), and a nucleic acid composition encoding a gRNA molecule of (a) and optionally a second, third and / or fourth gRNA molecule of (c).

[0129] In some embodiments, the contact step includes delivering to the cell a Cas9 molecule or Cas9-fusion protein as a protein or mRNA (b), the gRNA molecule as RNA (a), and optionally a second, third, and / or fourth gRNA molecule as RNA (c).

[0130] In some embodiments, contact includes delivering to the cell a gRNA molecule as RNA (a), a second, third and / or fourth gRNA molecule as RNA (c), and a nucleic acid composition encoding the Cas9 molecule or Cas9-fusion protein of said (b).

[0131] The subject matter disclosed in this invention also provides a method for treating, preventing, and / or alleviating the condition of a subject who has or may develop HSV-1 and / or HSV-2, for example by altering the structure (e.g., sequence) of a target nucleic acid of the subject, the method comprising contacting the subject (or cells derived from the subject) with:

[0132] (a) The gRNA molecules disclosed in this invention, for example, gRNA molecules targeting the RS1, RL2 or LAT genes;

[0133] (b) A Cas9 molecule (e.g., an eaCas9 molecule or an eiCas9 molecule) or a Cas9-fusion protein, such as the Cas9 molecule described in this invention; and

[0134] Optionally, (c)(i) a second gRNA molecule targeting the RS1, RL2, or LAT genes, such as the second gRNA molecule disclosed in this invention, and

[0135] Alternatively, (c)(ii) a third gRNA molecule, and still alternatively, (c)(iii) a fourth gRNA targeting the RL2 or LAT or RS1 gene, such as the third and fourth gRNA molecules disclosed in this invention.

[0136] In some implementations, the method includes introducing mutations at HSV RS1 targets, HSV RL2 targets, or HSV LAT targets, for example, via NHEJ.

[0137] In some implementations, the subject's cells are contacted in vivo with (e.g., via intravenous delivery), (a), (b), and optionally (c)(i), further optionally with (c)(ii), and still further optionally with (c)(iii).

[0138] In some embodiments, the contact step includes contacting the subject with a nucleic acid composition (e.g., the vector, AAV vector, or LV vector described in this invention), for example, a nucleic acid composition encoding at least one of (a), (b), and optionally (c)(i), further optionally (c)(ii), and still further optionally (c)(iii).

[0139] In some embodiments, the contact step includes delivering to the subject a Cas9 molecule or Cas9-fusion protein as a protein or mRNA (b), and a nucleic acid composition encoding (a) and optionally (c)(i), further optionally (c)(ii), and still further optionally (c)(iii).

[0140] In some embodiments, the contact step includes delivering to the subject a Cas9 molecule or Cas9-fusion protein as a protein or mRNA (b), a gRNA molecule as RNA (a), a second gRNA optionally as gRNA (c)(i), further optionally (c)(ii), and still further optionally (c)(iii) gRNA molecules.

[0141] In some embodiments, the contact step includes delivering to the subject a gRNA molecule as RNA (a), a second gRNA optionally as RNA (c)(i), further optionally (c)(ii), and still further optionally (c)(iii), and a nucleic acid composition encoding the Cas9 molecule or Cas9-fusion protein of (b).

[0142] When the method includes: (1) introducing a mutation at the HSV RS1 target, HSV RL2 target, or HSV LAT target via NHEJ or (2) knocking down the expression of one or more of the RS1, RL2, and / or LAT genes (e.g., by targeting the promoter region), the Cas9 molecule or Cas9-fusion protein of (b) and at least one gRNA molecule (e.g., the gRNA molecule of (a)) are included in the contact step.

[0143] The subject matter disclosed in this invention provides a reaction mixture comprising a gRNA molecule, a nucleic acid composition or a composition described in this invention, and cells (e.g., cells from a subject who has or may develop HSV-1 and / or HSV-2, or cells from a subject who would benefit from mutations at the HSV RL2 target, the HSV LAT target, or the HSV RS1 target).

[0144] The subject matter disclosed in this invention provides a kit comprising: (a) the gRNA molecule of this invention or a nucleic acid composition encoding the gRNA molecule, and one or more of the following:

[0145] (b) Cas9 molecules (e.g., eaCas9 molecules or eiCas9 molecules) or Cas9-fusion proteins, such as the Cas9 molecules described in this invention or nucleic acids or mRNA encoding said Cas9;

[0146] (c)(i) A second gRNA molecule, such as the second gRNA molecule described in this invention or a nucleic acid encoding (c)(i);

[0147] (c)(ii) A third gRNA molecule, such as the third gRNA molecule described in this invention or a nucleic acid encoding (c)(ii); or

[0148] (c)(iii) A fourth gRNA molecule, such as the fourth gRNA molecule described in this invention or a nucleic acid encoding (c)(iii). In some embodiments, the Cas9 molecule may be a Cas9 variant. For example, but not as a limitation, the Cas9 variant may be a Streptococcus pyogenes Cas9 variant or a Staphylococcus aureus Cas9 variant. In some embodiments, the Streptococcus pyogenes Cas9 variant is an EQR variant. In some embodiments, the Streptococcus pyogenes Cas9 variant is a VRER variant.

[0149] In some embodiments, the kit includes a nucleic acid encoding one or more of (a), (b), (c)(i), (c)(ii), and (c)(iii), for example, an AAV vector or an LV vector.

[0150] The subject matter disclosed in this invention provides a gRNA molecule (e.g., the gRNA molecule described herein) for treating, preventing, mitigating, or delaying the onset or progression of HSV-1 and / or HSV-2 infection in a subject, for example, according to the methods described herein for treating, preventing, mitigating, or delaying the onset or progression of HSV-1 and / or HSV-2 infection.

[0151] In some embodiments, the gRNA molecule is used in combination with a Cas9 molecule (e.g., an eaCas9 molecule or an eiCas9 molecule) or a Cas9-fusion protein (e.g., the Cas9 molecule described in this invention). For example, but not as a limitation, the Cas9 molecule or Cas9-fusion protein is a Cas9 variant. For example, but not as a limitation, the Cas9 variant may be a Streptococcus pyogenes Cas9 variant or a Staphylococcus aureus Cas9 variant. In some embodiments, the Streptococcus pyogenes Cas9 variant is an EQR variant. In some embodiments, the Streptococcus pyogenes Cas9 variant is a VRER variant. Additionally or alternatively, in some embodiments, the gRNA molecule is used in combination with a second, third, and / or fourth gRNA molecule, for example, the second, third, and / or fourth gRNA molecule described in this invention.

[0152] The subject matter disclosed in this invention provides for use in the preparation of medicaments for treating, preventing, alleviating, or delaying the onset or progression of HSV-1 and / or HSV-2 in subjects (e.g., the gRNA molecules described in this invention), for example, the methods for treating, preventing, alleviating, or delaying the onset or progression of HSV-1 and / or HSV-2 as described herein.

[0153] In some embodiments, the drug comprises a Cas9 molecule (e.g., an eaCas9 molecule or an eiCas9 molecule) or a Cas9-fusion protein (e.g., the Cas9 molecule described in this invention). Alternatively or additionally, in some embodiments, the drug comprises a second, third, and / or fourth gRNA molecule, for example, the second, third, and / or fourth gRNA molecule described in this invention. In some embodiments, the Cas9 molecule may be a Cas9 variant. For example, but not as a limitation, the Cas9 variant may be a Streptococcus pyogenes Cas9 variant or a Staphylococcus aureus Cas9 variant. In some embodiments, the Streptococcus pyogenes Cas9 variant is an EQR variant. In some embodiments, the Streptococcus pyogenes Cas9 variant is a VRER variant.

[0154] The gRNA molecules, genome editing systems, methods, compositions, reaction mixtures, and kits disclosed in this invention may also include controllable gRNA molecules, such as the controllable gRNA molecules disclosed in this invention.

[0155] Unless otherwise expressly defined, all technical and scientific terms used in this invention have the same meaning as understood by those skilled in the art in which this invention is described. Although methods and materials similar to or equivalent to those described and used in this invention may be used in the practice and testing of this invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned in this invention are incorporated herein by and through. Furthermore, materials, methods, and embodiments are merely exemplary and are not intended to be limiting.

[0156] Headings, including numeric and alphabetic headings and subheadings, are used for organization and presentation, not for limitation.

[0157] Other features and advantages of the invention will be apparent from the detailed description of the invention, the accompanying drawings and the claims. Brief description of the attached diagram

[0158] Figure 1A-1I These are representations of several exemplary gRNAs. Figure 1A Modular gRNA molecules with a double-stranded structure, partially derived from (or partially modeled on sequence) Streptococcus pyogenes (SEQ ID NO: 39 and 40, respectively, in order of appearance); Figure 1B A single gRNA molecule (SEQ ID NO:41) with a double-stranded structure derived from Streptococcus pyogenes was described. Figure 1C A single gRNA molecule (SEQ ID NO:42) with a double-stranded structure derived from Streptococcus pyogenes was described. Figure 1D A single gRNA molecule (SEQ ID NO:43) with a double-stranded structure derived from Streptococcus pyogenes was described. Figure 1E A single gRNA molecule (SEQ ID NO:44) with a double-stranded structure derived from Streptococcus pyogenes was described. Figure 1F Modular gRNA molecules with a double-stranded structure derived from Streptococcus thermophilus (SEQ ID NO:45 and 46 in order of appearance) were described. Figure 1G The alignment of modular gRNA molecules of Streptococcus pyogenes and Streptococcus thermophilus (SEQ ID NO: 39, 45, 47 and 46 in order of appearance) was depicted. Figure 1H-1I Another exemplary structure of a single gRNA molecule is depicted. Figure 1H An exemplary structure of a single gRNA molecule (SEQ ID NO:42) with a double-stranded structure derived from Streptococcus pyogenes is shown. Figure 1I An exemplary structure of a single gRNA molecule (SEQ ID NO:38) with a double-stranded structure derived from Staphylococcus aureus is shown.

[0159] Figure 2A-2G The alignment of the Cas9 sequence is depicted (Chylinski 2013). The N-terminal RuvC-like domain is boxed and indicated by "Y". The other two RuvC-like domains are boxed and indicated by "B". The HNH-like domain is boxed and indicated by "G". Sm: Streptococcus mutans (SEQ ID NO:1); Sp: Streptococcus pyogenes (SEQ ID NO:2); St: Streptococcus thermophilus (SEQ ID NO:4); and Li: Listeria monocytogenes (SEQ ID NO:5). The "motif" (SEQ ID NO:14) is a common sequence based on the four sequences. Conserved residues in all four sequences are represented by single-letter amino acid abbreviations; "*" indicates any amino acid found in the corresponding position of any of the four sequences; and "-" indicates absence.

[0160] Figures 3A-3B The comparison of the N-terminal RuvC-like domains from the Cas9 molecules (SEQ ID NO: 52-95, 120-123) disclosed in Chylinski 2013 is shown. Figure 3B The last line identified four highly conserved residues.

[0161] Figures 4A-4B The alignment of the N-terminal RuvC-like domain from the Cas9 molecule (SEQ ID NO:52-123) disclosed in Chylinski 2013 with sequence outliers removed is shown. Figure 4B The last line identified three highly conserved residues.

[0162] Figures 5A-5C The comparison of the HNH-like domains from the Cas9 molecule (SEQ ID NO:124-198) disclosed in Chylinski 2013 is shown. Figure 5C The last line identified conserved residues.

[0163] Figures 6A-6B The alignment of HNH-like domains from Cas9 molecules (SEQ ID NO: 124-141, 148, 149, 151-153, 162, 163, 166-174, 177-187, 194-198) disclosed in Chylinski 2013 with sequence outliers removed is shown. Figure 6B The last line identified three highly conserved residues.

[0164] Figure 7 The gRNA domain nomenclature using an exemplary gRNA sequence (SEQ ID NO:42) is shown.

[0165] Figure 8A and 8B A schematic representation of the domain organization of Streptococcus pyogenes Cas9 is provided. Figure 8A The organization of the Cas9 domain, including amino acid positions, is shown with reference to two types of Cas9 blades (Recognition (REC) blade and Nuclease (NUC) blade). Figure 8B The percentage homology of each domain in 83 Cas9 orthogonal homologs is shown.

[0166] Figure 9 This is a schematic diagram of the pAF025 plasmid map.

[0167] Figures 10A-10B The Cas9-mediated cleavage of the HSV-1 target sequence in plasmid pAF025 is shown. (A) Targeted knockdown of HSV1 RL2 / LAT is shown using the gRNAs listed in Table 18 (see Example 3). (B) Targeted knockdown of HSV1 RS1 is shown using the gRNAs listed in Table 19 (see Example 3). Invention Details

[0168] For the purpose of disclosure and not for limitation, the invention is detailed in the following sections:

[0169] 1. Definition

[0170] 2. Herpes simplex virus

[0171] 3. Methods for treating, preventing, and / or alleviating HSV-related ocular infections.

[0172] 4. Methods for altering the RS1, RL2, and / or LAT genes

[0173] 5. Guide RNA (gRNA) molecules

[0174] 6. Methods for designing gRNA

[0175] 7. Cas9 molecule

[0176] 8. Functional analysis of candidate molecules

[0177] 9. Genome editing methods

[0178] 10. Target cells

[0179] 11. Delivery, formulation and route of administration

[0180] 12. Modified nucleosides, nucleotides, and nucleic acids

[0181] 1. Definition

[0182] As used herein, the terms “about” or “approximately” refer to an acceptable margin of error for a particular value as determined by one of ordinary skill in the art, which may depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, “about” may mean within three or more standard deviations. Alternatively, “about” may mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Or, particularly with respect to biological systems or processes, the term may mean within an order of magnitude of the value, preferably within five times, more preferably within two times.

[0183] As used in this invention, a "genome editing system" refers to any system with RNA-guided DNA editing activity. The gene editing system disclosed in this invention comprises at least two components tuned from a naturally occurring CRISPR system: guide RNA (gRNA) and an RNA-guided nuclease. These two components form a complex capable of associating with a specific nucleic acid sequence in a cell and editing the DNA in or around that nucleic acid sequence, for example, by creating one or more single-strand breaks (SSBs or gaps), double-strand breaks (DSBs), and / or point mutations.

[0184] In various embodiments, the gene editing system may include (a) one or more Cas9 / gRNA complexes, and (b) separate Cas9 molecules and gRNAs capable of associating in cells to form one or more Cas9 / gRNA complexes. A genome editing system disclosed according to the present invention may be encoded by one or more nucleotides (e.g., RNA, DNA), said one or more nucleotides including coding sequences for Cas9 and / or gRNAs capable of associating to form Cas9 / gRNA complexes, said one or more nucleotides encoding said genome editing system may be delivered by the vectors described in the present invention.

[0185] In some embodiments, the genome editing system targets HSV viral genes selected from a group consisting of the RS1 gene, RL2 gene, and LAT gene. The genome editing system disclosed in this invention can be used to alter (knock out or knock down) one or more HSV viral genes, such as the RS1 gene, RL2 gene, and LAT gene.

[0186] In some embodiments, the genome editing system targets the RS1 gene. In some embodiments, the RS1 gene is the human RS1 gene. In some embodiments, the genome editing system targets the RL2 gene. In some embodiments, the RL2 gene is the human RL2 gene. In some embodiments, the genome editing system targets the LAT gene. In some embodiments, the LAT gene is the human LAT gene. In some embodiments, the genome editing system targets two or three of the RS1, RL2, and LAT genes.

[0187] In some embodiments, the gene editing system targeting the RS1 gene includes a first gRNA molecule or a polynucleotide encoding thereas and at least one Cas9 molecule or a polynucleotide encoding thereas, wherein the first gRNA molecule includes a target domain complementary to a target domain (also referred to as a “target sequence”) in the RS1 gene. In some embodiments, the genome editing system targeting the RS1 gene further includes a second gRNA molecule, which includes a target domain complementary to a second target domain in the RS1 gene or a polynucleotide encoding thereas. The genome editing system targeting the RS1 gene may also include third and fourth gRNA molecules targeting the RS1 gene.

[0188] In some embodiments, the gene editing system targeting the RL2 gene includes a first gRNA molecule or a polynucleotide encoding thereas and at least one Cas9 molecule or a polynucleotide encoding thereas, wherein the first gRNA molecule includes a target domain complementary to a target domain in the RL2 gene. In some embodiments, the genome editing system targeting the RL2 gene further includes a second gRNA molecule, which includes a target domain complementary to a second target domain in the RL2 gene or a polynucleotide encoding thereas. The genome editing system targeting the RL2 gene may also include third and fourth gRNA molecules targeting the RL2 gene.

[0189] In some embodiments, the gene editing system targeting the LAT gene includes a first gRNA molecule or a polynucleotide encoding thereof and at least one Cas9 molecule or a polynucleotide encoding thereof, wherein the first gRNA molecule includes a target domain complementary to a target domain in the LAT gene. In some embodiments, the genome editing system targeting the LAT gene further includes a second gRNA molecule, which includes a target domain complementary to a second target domain in the LAT gene or a polynucleotide encoding thereof. The genome editing system targeting the LAT gene may also include third and fourth gRNA molecules targeting the LAT gene.

[0190] In some embodiments, the genome editing system is implemented in cells or in vitro or in vivo. In some embodiments, the genome editing system is used in pharmaceuticals, such as those for modifying one or more target genes (e.g., RS1, RL2, and / or LAT genes) or for treating, preventing, and / or alleviating HSV infection (HSV-1 or HSV-2 infection). In some embodiments, the genome editing system is used for treatment.

[0191] As used in this invention, "target gene" refers to any nucleotide sequence encoding a known or presumed gene product. In some embodiments, the target gene is an HSV viral gene. As used in this invention, "HSV viral gene" refers to the (HSV-1 or HSV-2)RS1 gene, the (HSV-1 or HSV-2)RL2 gene, or the (HSV-1 or HSV-2)LAT gene.

[0192] The term "HSV1 RS1 target knockout site" used in this invention refers to a location within the RS1 gene of HSV1. If this location is altered by NHEJ-mediated changes, the expression of the functional RS1 gene product is reduced or eliminated. In some embodiments, the location is within the coding region of the RS1 gene (e.g., an early coding region). In other embodiments, the location is within the non-coding region of the RS1 gene.

[0193] The term "HSV2 RS1 target knockout site" used in this invention refers to a location within the RS1 gene of HSV2. If this location is altered by NHEJ-mediated changes, the expression of the functional RS1 gene product is reduced or eliminated. In some embodiments, the location is within the coding region of the RS1 gene (e.g., an early coding region). In other embodiments, the location is within the non-coding region of the RS1 gene.

[0194] The term "HSV RS1 target knockout site" used in this invention refers to a location within the RS1 gene of an HSV (e.g., HSV-1 or HSV-2) that, if altered by NHEJ-mediated changes, results in a reduction or elimination of the expression of the functional RS1 gene product. In some embodiments, the location is within the coding region of the RS1 gene (e.g., an early coding region). In other embodiments, the location is within the non-coding region of the RS1 gene.

[0195] The term "HSV1 RS1 target knockdown location" used in this invention refers to a location within the RS1 gene of HSV1. If this location is targeted by the eiCas9 or eiCas9-fusion protein described in this invention, the expression of the functional RS1 gene product is reduced or eliminated. In some embodiments, transcription is reduced or eliminated. In some embodiments, the location is situated within the promoter region of the RS1 gene (e.g., a location within the promoter region of the RS1 gene targeted by eiCas9 or the eiCas9-fusion protein).

[0196] The term "HSV2 RS1 target knockdown location" used in this invention refers to a location within the RS1 gene of HSV2. If this location is targeted by the eiCas9 or eiCas9-fusion protein described in this invention, the expression of the functional RS1 gene product is reduced or eliminated. In some embodiments, transcription is reduced or eliminated. In some embodiments, the location is situated within the promoter region of the RS1 gene (e.g., a location within the promoter region of the RS1 gene targeted by eiCas9 or the eiCas9-fusion protein).

[0197] The term "HSV RS1 target knockdown location" used in this invention refers to a location within the RS1 gene of an HSV (e.g., HSV-1 or HSV-2) that, if targeted by the eiCas9 or eiCas9-fusion protein described in this invention, results in a reduction or elimination of the expression of the functional RS1 gene product. In some embodiments, transcription is reduced or eliminated. In some embodiments, the location is situated within the promoter region of the RS1 gene (e.g., a location within the promoter region of the RS1 gene targeted by the eiCas9 or eiCas9-fusion protein).

[0198] The “HSV RS1 target” used in this invention includes HSV RS1 target knockdown locations and / or HSV RS1 target knockout locations.

[0199] The term "HSV1 RL2 target knockout site" used in this invention refers to a location within the RL2 gene of HSV1. If this location is altered by NHEJ-mediated changes, the expression of the functional RL2 gene product is reduced or eliminated. In some embodiments, the location is within the coding region of the RL2 gene (e.g., an early coding region). In other embodiments, the location is within the non-coding region of the RL2 gene.

[0200] The term "HSV2 RL2 target knockout site" used in this invention refers to a location within the RL2 gene of HSV2. If this location is altered by NHEJ-mediated changes, the expression of the functional RL2 gene product is reduced or eliminated. In some embodiments, the location is within the coding region of the RL2 gene (e.g., an early coding region). In other embodiments, the location is within the non-coding region of the RL2 gene.

[0201] The term "HSV RL2 target knockout site" used in this invention refers to a location within the RL2 gene of an HSV (e.g., HSV-1 or HSV-2) that, if altered by NHEJ-mediated changes, results in a reduction or elimination of the expression of the functional RL2 gene product. In some embodiments, the location is within the coding region of the RL2 gene (e.g., an early coding region). In other embodiments, the location is within the non-coding region of the RL2 gene.

[0202] The term "HSV1 RL2 target knockdown location" used in this invention refers to a location within the RL2 gene of HSV1. If this location is targeted by the eiCas9 or eiCas9-fusion protein described in this invention, the expression of the functional RL2 gene product is reduced or eliminated. In some embodiments, transcription is reduced or eliminated. In some embodiments, the location is situated within the promoter region of the RL2 gene (e.g., a location within the promoter region of the RL2 gene targeted by eiCas9 or the eiCas9-fusion protein).

[0203] The term "HSV2 RL2 target knockdown location" used in this invention refers to a location within the RL2 gene of HSV2. If this location is targeted by the eiCas9 or eiCas9-fusion protein described in this invention, the expression of the functional RL2 gene product is reduced or eliminated. In some embodiments, transcription is reduced or eliminated. In some embodiments, the location is situated within the promoter region of the RL2 gene (e.g., a location within the promoter region of the RL2 gene targeted by eiCas9 or the eiCas9-fusion protein).

[0204] The term "HSV RL2 target knockdown location" used in this invention refers to a location within the RL2 gene of an HSV (e.g., HSV-1 or HSV-2) that, if targeted by the eiCas9 or eiCas9-fusion protein described in this invention, results in a reduction or elimination of the expression of the functional RL2 gene product. In some embodiments, transcription is reduced or eliminated. In some embodiments, the location is situated within the promoter region of the RL2 gene (e.g., a location within the promoter region of the RL2 gene targeted by the eiCas9 or eiCas9-fusion protein).

[0205] The “HSV RL2 target location” used in this invention includes the HSV RL2 target knockdown location and / or the HSV RL2 target knockout location.

[0206] The term "HSV1 LAT target knockout site" used in this invention refers to a location within the HSV1 LAT gene where, if this location is altered by NHEJ-mediated changes, the expression of the functional LAT gene product is reduced or eliminated. In some embodiments, the location is situated within the coding region of the LAT gene (e.g., an early coding region). In other embodiments, the location is situated within the non-coding region of the LAT gene.

[0207] The term "HSV2 LAT target knockout site" used in this invention refers to a location within the LAT gene of HSV2. If this location is altered by NHEJ-mediated changes, the expression of the functional LAT gene product is reduced or eliminated. In some embodiments, the location is within the coding region of the LAT gene (e.g., an early coding region). In other embodiments, the location is within the non-coding region of the LAT gene.

[0208] The term "HSV LAT target knockout site" used in this invention refers to a location within the LAT gene of HSV (e.g., HSV-1 or HSV-2) that, if altered by NHEJ-mediated changes, results in a reduction or elimination of the expression of the functional LAT gene product. In some embodiments, the location is within the coding region of the LAT gene (e.g., an early coding region). In other embodiments, the location is within the non-coding region of the LAT gene.

[0209] The term "HSV1 LAT target knockdown location" used in this invention refers to a location within the HSV1 LAT gene where, if this location is targeted by the eiCas9 or eiCas9-fusion protein described in this invention, the expression of the functional LAT gene product is reduced or eliminated. In some embodiments, transcription is reduced or eliminated. In some embodiments, the location is situated within the promoter region of the LAT gene (e.g., a location within the promoter region of the LAT gene targeted by eiCas9 or the eiCas9-fusion protein).

[0210] The term "HSV2 LAT target knockdown location" used in this invention refers to a location within the LAT gene of HSV2. If this location is targeted by the eiCas9 or eiCas9-fusion protein described in this invention, the expression of the functional LAT gene product is reduced or eliminated. In some embodiments, transcription is reduced or eliminated. In some embodiments, the location is situated within the promoter region of the LAT gene (e.g., a location within the promoter region of the LAT gene targeted by eiCas9 or the eiCas9-fusion protein).

[0211] The term "HSV LAT target knockdown location" used in this invention refers to a location within the LAT gene of an HSV (e.g., HSV-1 or HSV-2) that, if targeted by the eiCas9 or eiCas9-fusion protein described in this invention, results in a reduction or elimination of the expression of the functional LAT gene product. In some embodiments, transcription is reduced or eliminated. In some embodiments, the location is situated within the promoter region of the LAT gene (e.g., a location within the promoter region of the LAT gene targeted by eiCas9 or the eiCas9-fusion protein).

[0212] The “HSV LAT target location” used in this invention includes the HSV LAT target knockdown location and / or the HSV LAT target knockout location.

[0213] As used in this article, "domain" is a segment used to describe a protein or nucleic acid. Unless otherwise specified, a domain is not required to have any specific functional properties.

[0214] The calculation of homology or sequence identity (these terms are used interchangeably throughout this document) between two sequences is performed as follows. These sequences are aligned for optimal comparison purposes (e.g., for optimal alignment, vacancies may be introduced in one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences may be disregarded for comparison purposes). The optimal alignment is determined as the best score using the GAP procedure in the GCG software package with a Blossum 62 scoring matrix (where the vacancy penalty is 12, the vacancy extension penalty is 4, and the frameshift vacancy penalty is 5). The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. The molecules are identical at that position when a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence. The percentage identity between two sequences is a function of the number of identical positions shared by the sequences.

[0215] The term "dominant gRNA molecule" as used in this invention refers to a gRNA molecule comprising a targeting domain complementary to a target domain on a nucleic acid comprising a sequence encoding a component of a CRISPR / Cas system introduced into a cell or subject. The dominant gRNA does not target endogenous cell or subject sequences. In some embodiments, the dominant gRNA molecule comprises a targeting domain complementary to a target sequence located on: (a) a nucleic acid encoding a Cas9 molecule; (b) a nucleic acid encoding a gRNA comprising a targeting domain targeting RS1, RL2, or LAT genes (target gene gRNA); or located on more than one nucleic acid encoding a CRISPR / Cas component (both (a) and (b)). In some embodiments, the nucleic acid molecule encoding a CRISPR / Cas component (e.g., encoding a Cas9 molecule or a target gene gRNA) comprises more than one target domain complementary to the dominant gRNA targeting domain. In some embodiments, the dominant gRNA molecule complexes with the Cas9 molecule and causes Cas9-mediated inactivation of the targeted nucleic acid (e.g., by cleavage or by binding to the nucleic acid) and results in the cessation or reduction of the production of CRISPR / Cas system components. In some embodiments, the Cas9 molecule forms two complexes: one complex comprising the Cas9 molecule and the target gene gRNA, which can alter the RS1, RL2, or LAT gene; and another complex comprising the Cas9 molecule and the dominant gRNA molecule, which can be used to prevent further production of CRISPR / Cas system components (e.g., the Cas9 molecule or the target gene gRNA molecule). In some embodiments, the dominant gRNA molecule / Cas9 molecule complex binds to or promotes the cleavage of a control region sequence, which is operatively linked to a sequence encoding the Cas9 molecule, a transcriptional region, an exon, or an intron. In some embodiments, the dominant gRNA molecule / Cas9 molecule complex binds to or promotes the cleavage of a control region sequence, which is operatively linked to the gRNA molecule or a sequence encoding the gRNA molecule. In some embodiments, a dominant gRNA (e.g., a Cas9-targeting dominant gRNA molecule or a target gene gRNA-targeting control gRNA molecule) restricts the gene targeting mediated by the Cas9 / target gene gRNA complex. In some embodiments, the dominant gRNA imposes temporal, level-based, or other restrictions on the activity of the Cas9 / target gene gRNA complex. In some embodiments, the dominant gRNA reduces off-target or other unwanted activities.In some implementations, dominant gRNA molecules inhibit (e.g., completely or substantially completely inhibit) the production of components of the Cas9 system and thereby limit or dominate its activity.

[0216] As used in this invention, a "regulator" refers to an entity, such as a drug, that can alter the activity (e.g., enzyme activity, transcriptional activity, or translational activity), quantity, distribution, or structure of a target molecule or gene sequence. In some embodiments, regulation includes cleavage, e.g., the breaking of covalent or non-covalent bonds, or the formation of covalent or non-covalent bonds, e.g., moiety attachment to the target molecule. In some embodiments, the regulator alters the three-dimensional, secondary, tertiary, or quaternary structure of the target molecule. The regulator can increase, decrease, activate, or eliminate target activity.

[0217] In this invention, "macromolecule" refers to a molecule with a molecular weight of at least 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kDa. Macromolecules include proteins, polypeptides, nucleic acids, biological agents, and carbohydrates.

[0218] As used in this invention, "polypeptide" refers to a polymer of amino acids having fewer than 100 amino acid residues. In some embodiments, it has fewer than 50, 20, or 10 amino acid residues.

[0219] As used herein, "Cas9 molecule" or "Cas9 peptide" refers to a molecule or peptide that can interact with a gRNA molecule and, together with the gRNA molecule, localize to a site including a target domain (and in some embodiments, a PAM sequence) (also referred to as the "target sequence"). Cas9 molecules and Cas9 peptides include naturally occurring Cas9 molecules and Cas9 peptides, as well as engineered, altered, or modified Cas9 molecules or Cas9 peptides that differ from a reference sequence (e.g., the most similar naturally occurring Cas9 molecule) by, for example, at least one amino acid residue.

[0220] In some embodiments, the Cas9 molecule is wild-type *Streptococcus pyogenes* Cas9 that recognizes the NGG PAM sequence. In some embodiments, the Cas9 molecule is a *Streptococcus pyogenes* Cas9 EQR variant that recognizes the NGAG PAM, NGCG PAM, NGGG PAM, NGTG PAM, NGAA PAM, NGAT PAM, or NGAC PAM sequences. In some embodiments, the Cas9 molecule is a *Streptococcus pyogenes* Cas9 VRER variant that recognizes the NGCG PAM, NGCA PAM, NGCT PAM, or NGCC PAM sequences. In some embodiments, the Cas9 molecule is wild-type *Staphylococcus aureus* Cas9 that recognizes the NNGRRT PAM or NNGRRV PAM sequences.

[0221] As used in this invention, a "reference molecule" refers to a molecule to which a modified or candidate molecule is compared. For example, a reference Cas9 molecule is a Cas9 molecule to which a modified or candidate Cas9 molecule is compared. Similarly, a reference gRNA is a gRNA molecule to which a modified or candidate gRNA molecule is compared. Modified or candidate molecules can be compared to a reference molecule based on sequence (e.g., the modified or candidate molecule may have X% sequence identity or homology with the reference molecule) or activity (e.g., the modified or candidate molecule may have X% of the activity of the reference molecule). For example, in the case where the reference molecule is a Cas9 molecule, the modified or candidate molecule may be characterized as having no more than 10% of the nuclease activity of the reference Cas9 molecule. Examples of reference Cas9 molecules include naturally occurring, unmodified Cas9 molecules, such as naturally occurring Cas9 molecules from Streptococcus pyogenes, Staphylococcus aureus, or Neisseria meningitidis. In some embodiments, the reference Cas9 molecule is a naturally occurring Cas9 molecule that has the closest sequence identity or homology to the modified or candidate Cas9 molecule to which it is compared. In some implementations, the reference Cas9 molecule is a parent molecule having a naturally occurring or known sequence, to which mutations are made to achieve a modified or candidate Cas9 molecule.

[0222] The use of "substitution" or "alternative" in relation to the modification of molecules in this invention does not require method limitation, but merely indicates that the substitution entity exists.

[0223] The term "small molecule" as used in this invention refers to compounds with a molecular weight of less than about 2 kD, such as less than about 2 kD, less than about 1.5 kD, less than about 1 kD, or less than about 0.75 kD.

[0224] As used in this invention, "subject" can refer to a human or a non-human animal. The term includes, but is not limited to, mammals (e.g., humans, other primates, pigs, rodents (e.g., mice and rats or hamsters), rabbits, guinea pigs, cows, horses, cats, dogs, sheep, and goats). In some embodiments, the subject is a human. In other embodiments, the subject is poultry.

[0225] The term "treatment" as used in this invention refers to the treatment of diseases in mammals, such as humans, including (a) suppressing the disease, i.e., preventing or stopping its development or progression; (b) alleviating the disease, i.e. causing the disease state to subside; (c) alleviating one or more symptoms of the disease; and (d) curing the disease.

[0226] The term “preventing” as used in this invention refers to the prevention of diseases in mammals, such as humans, including (a) avoiding or eliminating diseases; (b) influencing disease predisposition; and (c) preventing or delaying the onset of at least one symptom of a disease.

[0227] As used herein, “X” in the context of an amino acid sequence refers to any amino acid (e.g., any one of the twenty natural amino acids) unless otherwise stated.

[0228] 2. Herpes simplex virus

[0229] Herpes simplex virus (HSV) is divided into at least two types: herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2). HSV-1 and HSV-2 are also known as human herpesvirus 1 (HHV-1) and human herpesvirus (HHV-2), respectively.

[0230] The structure of herpesviruses consists of a relatively large double-stranded linear DNA genome enclosed in an icosahedral protein cage (capsid), which is then encased in a lipid bilayer called the envelope. The envelope attaches to the capsid via the membrane. The complete particle is called a virion (Mettenleiter et al., (2006) Curr. Opin. Microbiol. 9(4):423-429). HSV-1 and HSV-2 each contain at least 74 genes (or open reading frames, ORFs) or even up to 84 unique protein-coding genes in their genomes, which are composed of 94 putative ORFs (McGeoch et al., (2006) VirusRes. 117(1):90–104; Rajcáni et al., (2004) Virus Genes 28(3):293-310). These genes encode various proteins involved in the formation of the viral capsid, membrane, and envelope, as well as those controlling viral replication and infectivity.

[0231] The genomes of HSV-1 and HSV-2 are complex and comprise two distinct regions: the long unique region (UL) and the short unique region (US), each containing multiple viral genes. Immediately early genes encode proteins that regulate the expression of early and late viral genes, such as those involved in DNA replication and the production of certain envelope glycoproteins. Late genes encode proteins that form viral particles, such as those involved in viral particle formation. Transcription of HSV genes is catalyzed by RNA polymerase II in the infected host (McGeoch et al. (2006) Virus Res. 117(1):90-104).

[0232] HSV entry into host cells involves the interaction of several glycoproteins on the viral envelope (e.g., glycoprotein B (gB), glycoprotein C (gC), glycoprotein D (gD), glycoprotein H (gH), and glycoprotein L (gL)) with receptors on the host cell surface (e.g., herpesvirus entry mediator (HVEM), Nectin-1, or 3-O-sulfated heparan sulfate)). Upon binding to a specific receptor on the cell surface, the envelope fuses with the host cell membrane, creating a pore through which the virus enters the host cell. The virus can also be internalized after receptor binding, and fusion can occur in endosomes. After entering the cytoplasm, the viral capsid is transported to the nucleus. Once attached to the nucleus at the nuclear entrance pore, the capsid expels its DNA contents through the capsid entrance. Following cell infection, a cascade of herpesvirus proteins is produced, such as immediate early, early, and late proteins.

[0233] Herpes simplex virus can persist in a quiescent but persistent form, known as latent infection. During latent infection in cells, HSV expresses latent-associated transcript (LAT) RNA. LAT modulates the host cell genome and interferes with natural cell death mechanisms. By maintaining host cells, LAT expression preserves a viral reservoir, leading to subsequent, usually symptomatic, periodic relapses or non-latent “outbreaks.” Regardless of whether a relapse is symptomatic, viral shedding occurs to generate further infection. Herpesvirus DNA contains a gene encoding ICP4, which is a transactivator of genes associated with lytic infection (Pinnoji et al. (2007) Virol. J. 4:56). Human neuronal protein neuron restriction silencing factor (NRSF) or human repressive element silencing transcription factor (REST) ​​can bind to elements surrounding the ICP4 gene and cause histone deacetylation, thereby preventing transcription initiation from this gene and subsequently preventing transcription of other viral genes involved in the cleavage cycle (Pinnoji et al. (2007) Virol. J. 4: 56; Bedadala et al. (2007) Cell Res. 17(6): 546-555). The inhibition of ICP4 protein synthesis can be reversed by the viral protein ICP0, which causes NRSF to dissociate from the ICP4 gene, thereby preventing the silencing of viral DNA (Roizman et al. (2005) Cell Cycle 4(8): 1019-21).

[0234] 2.1 HSV infection

[0235] Herpes simplex virus enters the host by infecting epithelial cells in the skin and mucous membranes. Most commonly, HSV-1 enters the host through infection of oropharyngeal epithelial cells (including the epithelial cells of the mouth, lips, and nose). Most commonly, HSV-2 enters the host through infection of epithelial cells in the anogenital region (including the epithelium of the genitals and anus). However, HSV-1 primarily infects the anogenital region, while HSV-2 primarily infects the oropharynx.

[0236] HSV-1 causes intermittent pain in the mouth and mucous membranes. It is a ubiquitous and highly contagious pathogen. Initial infection with HSV-1 typically causes painful blisters on the lips and oral mucosa.

[0237] HSV-2 is a sexually transmitted virus. It is commonly known as genital herpes. Initial HSV-2 infection typically causes painful blisters in the genital area. The disease can cause recurrent viral responses throughout life. It is highly contagious and increases the risk of HIV infection, especially in patients with active lesions.

[0238] HSV-1 and HSV-2 infections persist throughout a host's life. During primary infection, the virus typically infects cells in the oropharyngeal and anogenital regions, causing painful vesicles in the affected areas. Reactivation of HSV infection usually occurs in the oropharyngeal or anogenital regions. However, reactivation of the eye and central nervous system is the most serious and detrimental HSV manifestation, as it can lead to blindness and permanent neurological impairment, respectively. Primary and reactivated infections can result in permanent neurological sequelae and blindness. HSV-2 also increases the risk of developing HIV in individuals. There is a considerable need for methods to treat, prevent, and / or reduce HSV-1 and / or HSV-2 infection.

[0239] Herpes simplex virus (HSV) produces immediate early genes within epithelial cells, encoding enzymes and binding proteins required for viral synthesis. Following primary infection, the virus is transported retrogradely from sensory nerve axons to the dorsal root ganglia (DRGs). HSV-1 primarily reaches the trigeminal DRG, but can spread to other sensory ganglia depending on the primary site of infection. HSV-2 primarily reaches the sensory DRG located in the sacrum, but can reach other sensory ganglia depending on the primary site of infection. In the DRG, the virus establishes a latent infection. This latent infection persists throughout the host's life. Within the DRG cells, the virus uncapsulates, viral DNA is transported to the nucleus, and key viral RNAs associated with the latent period are transcribed (including LAT RNA).

[0240] During primary infection, subjects typically experience painful blisters in the oral or anogenital area for 4–15 days. The most common areas of pain include the lips, gums, and nasal mucosa in primary HSV-1 infection. Less commonly, primary HSV-1 infection may involve the anogenital area. Primary HSV-2 infection is most common in the anogenital area, including the vagina, labia, cervix, penis, scrotum, anus, and skin around the thighs. Less commonly, primary HSV-2 infection may involve the oropharynx. Very rarely, primary HSV-1 and HSV-2 infections may involve the eyes, central nervous system, fingers, and nail beds (herpes vitiligo). HSV-1 infection is primarily transmitted through saliva and / or sexual contact. HSV-2 infection is primarily transmitted through sexual contact, but can also be transmitted through saliva. Blisters from HSV infection may rupture, releasing a highly infectious, clear fluid. Primary infection is often accompanied by flu-like illness, including fever, chills, and muscle aches.

[0241] Host immune defenses are crucial against HSV infection. CD4+ T cells and CD8+ cells are responsible for recognizing and clearing the pathogen. Subjects with impaired T cell responses, including HIV patients, organ transplant recipients receiving immunosuppressants, and newborns whose immune systems are still developing, are more susceptible to the most severe manifestations of HSV-1 and HSV-2 infection.

[0242] Reactivation of latent infections is usually less severe and may be shorter in duration. Reactivation of HSV-1 infection most commonly affects the oral region, but can also affect other areas, including the anogenital region, eyes, central nervous system (CNS), nails, and pharynx. Reactivation of HSV-2 infection typically affects the anogenital region, but can also affect other areas, including the oral region, eyes, central nervous system (CNS), nails, and pharynx. Reactivation of HSV-1 or HSV-2 infection can lead to ophthalmic conditions, including keratitis (epithelial keratitis, stromal keratitis, and discoid keratitis). Generally, ophthalmic manifestations of HSV-1 and HSV-2 include pain, tearing, redness of the eye, and light sensitivity. Most HSV-related ocular infections resolve without permanent vision loss. However, ocular herpes infections rarely lead to scarring, secondary bacterial infections, and rarely blindness. Reactivation of HSV-1 or HSV-2 infection can also cause retinitis. HSV-related retinitis is rare but severe and carries a high risk of permanent blindness.

[0243] Newborns are at high risk of severe HSV-1 and HSV-2 infections. These diseases are transmitted from mother to fetus during delivery. The chance of mother-to-fetal transmission is highest if the mother has a primary HSV infection during pregnancy. The incidence of neonatal herpes is approximately 4–30 per 100,000 newborns. Newborns can develop severe HSV-1 or HSV-2 encephalitis and / or meningitis. Despite immediate antiviral treatment, the incidence of permanent neurological sequelae in newborns infected with HSV-1 or HSV-2 is significant. In a study of infants with HSV encephalitis or meningitis treated with high-dose antiviral therapy, a mortality rate of 4% was found, with 69% of survivors experiencing permanent neurological sequelae (Kimberlin et al., Pediatrics, 2001; 108:230-238).

[0244] Primary HSV-1 and HSV-2 infections can be treated with antiviral therapies, including acyclovir, valacyclovir, and famciclovir. These treatments have been shown to reduce viral shedding, pain, and improve wound healing time. Reactivation of latent infection may resolve without treatment (potentially self-limiting) or may be treated with antiviral therapies. Treatment is primarily administered during acute infection. There are no curative or prophylactic treatments. Treatment may be given prophylactically in certain circumstances, including during delivery of a mother with a recent HSV-1 or HSV-2 infection or reactivation.

[0245] There is no effective treatment to prevent HSV-1 or HSV-2 infection. Antiviral treatment and condom use during active infection can reduce the rate of transmission by approximately 50%.

[0246] Human immunodeficiency virus-1 (HIV-1) infection rates were significantly increased in HSV-2 seropositive subjects. The risk of HIV-1 infection was three times higher in HSV-2 patients. Antiviral drugs had no effect on reducing the risk of HIV infection.

[0247] 2.2 HSV-related eye diseases

[0248] HSV infections, such as HSV-1 and / or HSV-2 infections of the eye (primary or reactivated), are known as HSV-associated eye disease. HSV-associated eye disease most commonly causes anterior chamber infection, called keratitis, stromal keratitis, and / or discoid keratitis. HSV-associated eye disease may less rarely cause posterior chamber infection, called retinitis. HSV-1 keratitis is intensely painful and unpleasant. It rarely leads to scarring, secondary infection by bacterial pathogens, and rarely results in blindness. HSV-associated retinitis is a rare manifestation of HSV-associated eye disease but carries a higher risk of permanent vision impairment.

[0249] Reactivation of the infection occurs in the eye by antegrade transport of the virus from the trigeminal ganglion along the ophthalmic branch of the trigeminal nerve (the fifth cranial nerve). Reactivation can also occur within the cornea. Latency within the trigeminal ganglion is established through one of two mechanisms. First, HSV-1 or HSV-2 can be transported retrogradely from the eye (after ocular infection) along the trigeminal nerve. Alternatively, it can spread to the trigeminal ganglion via hematogenous dissemination after infecting the oral mucosa, genital site, or other extraocular sites. After establishing latent infection in the trigeminal ganglion, the virus can enter the eye at any time, especially in immunocompromised hosts, by antegrade travel along the trigeminal nerve and re-establishing infection.

[0250] When ocular herpesviruses affect the posterior chamber of the eye, they can cause retinitis. In adults, HSV-1 is the cause of most cases of HSV-retinitis (Pepose et al., Ophthalmic Infections and Immunology, 1996; Mosby 1155–1168). In newborns and children, HSV-2 is the cause of most cases of HSV-retinitis (Pepose et al., Ophthalmic Infections and Immunology, 1996; Mosby 1155–1168). HSV-associated retinitis can lead to acute retinal necrosis (ARN), which can destroy the retina within 2 weeks without treatment (Banerjee and Rouse, Human Herpesviruses, 2007; Cambridge University Press, Chapter 35). Even with treatment, the risk of permanent vision impairment after ARN is greater than 50% (Roy et al., Ophthalmic Immunology and Inflammation 2014; 22(3): 170–174).

[0251] Keratitis is the most common form of ocular herpes simplex. HSV keratitis can manifest as dendritic keratitis, stromal keratitis, blepharitis, and conjunctivitis. HSV-1 is the cause of the majority of HSV-related keratitis cases, accounting for 58% of cases (Dawson et al., Review of Ophthalmology, 1976; 21(2):121-135). HSV-2 accounts for the remaining cases of HSV-related keratitis, approximately 42%. In the United States, there are approximately 48,000 cases of recurrent or primary HSV-related keratitis each year (Liesegang et al., 1989; 107(8):1155-1159). In all cases of HSV-related keratitis, approximately 1.5–3% of subjects experience severe permanent visual impairment (Wilhelmus et al., Archives of Ophthalmology, 1981; 99(9):1578-82). The risk of permanent visual impairment due to HSV-related ophthalmopathy increases with the number of ocular HSV reactivations.

[0252] In general, stromal keratitis accounts for about 15% of all keratitis cases and is associated with the highest risk of permanent visual impairment from keratitis. Stromal keratitis leads to scarring and irregular astigmatism. A prior ocular HSV infection increases the risk of developing a stromal infection, meaning that subjects with a previous ocular HSV infection have an increased risk of permanent visual impairment upon reactivation. In children, stromal keratitis accounts for 60% of all keratitis cases. Therefore, children are particularly vulnerable to permanent visual impairment from HSV-associated keratitis. A retrospective study in the United States from 1950 to 1982 found approximately 2.6 new or recurrent cases of stromal keratitis per 100,000 person-years, or approximately 8,000 cases per year (Liesegang et al., 1989; 107(8):1155-1159). A recent study conducted in France in 2002 estimated the incidence of new or recurrent stromal keratitis to be 9.6 per 100,000 (Labetoulle et al., Ophthalmology, 2005; 112(5):888-895). In developed countries, the incidence of HSV-associated keratitis may be increasing (Farooq and Shukla, 2012; Ophthalmology Review, 57(5):448-462).

[0253] The genome editing systems, compositions, and methods described in this invention can be used to treat, prevent, and / or reduce ocular infections of HSV-1 and / or HSV-2, including but not limited to HSV-1 stromal keratitis, HSV-1 dendritic keratitis, HSV-1 blepharitis, HSV-1 conjunctivitis, HSV-1 retinitis, HSV-2 stromal keratitis, HSV-2 dendritic keratitis, HSV-2 blepharitis, HSV-2 conjunctivitis, and HSV-2 retinitis.

[0254] 3. Methods for treating, preventing, and / or alleviating HSV-related ocular infections.

[0255] This invention discloses pathways for treating, preventing, and / or reducing HSV-related eye infections using the methods, genome editing systems, and compositions described herein. HSV-related eye infections may be caused by HSV-1 and / or HSV-2 infections. For example, and not as a limitation, the methods, genome editing systems, and compositions disclosed herein can be used to treat, prevent, and / or reduce HSV-1 infection, HSV-2 infection, or both HSV-1 and HSV-2 infection.

[0256] The RS1, RL2, and LAT genes in HSV-1 and HSV-2 are involved in viral infection, replication, assembly, and the maintenance of latency and viral reactivation. Knocking out or downsetting any of these individual or combined genes reduces HSV-1 and / or HSV-2 infection. Because HSV-1 or HSV-2 viruses establish latency in dispersed local areas throughout the body, they are well-suited for local delivery of ineffective treatments to these latent areas. Targeted knockout in dispersed areas (e.g., the dorsal root ganglion of the trigeminal nerve, cornea, dorsal root ganglion of the neck, or dorsal root ganglion of the sacrum) can reduce or eliminate latent infection by inactivating HSV-1 and / or HSV-2 viruses.

[0257] This invention describes methods for treating, preventing, and / or reducing HSV-1 and / or HSV-2 infection by knocking out or knocking down viral genes. The methods described herein involve knocking out or knocking down the following genes encoding HSV-1 and / or HSV-2: RL2, LAT, and RS1, or any combination thereof (e.g., any single gene, such as RL2, such as LAT, such as RS1; or any two genes, such as RL2 and LAT, such as RL2 and RS1, such as RS1 and LAT; or three genes). When two alteration events (e.g., knockdown or knockout of RS1, RL2, and / or LAT gene expression) are present, the two alteration events may occur sequentially or simultaneously. In some embodiments, the knockout of RS1, RL2, and / or LAT genes occurs prior to the knockdown of RS1, RL2, and / or LAT genes. In some embodiments, the knockout of RS1, RL2, and / or LAT genes occurs simultaneously with the knockdown of RS1, RL2, and / or LAT genes. In some implementations, knockout of the RS1, RL2, and / or LAT genes occurs after knockdown of the RS1, RL2, and / or LAT genes. In some implementations, the effects of the changes are synergistic.

[0258] RL2 encodes the gene ICP0, a 775-amino acid protein that acts as a transactivator of gene expression. The RL2 gene is one of five immediate early genes expressed by herpesviruses. ICP0 is involved in the activation of delayed early and late gene expression (Lees-Miller et al., 1996, Journal of Virology, 70(11):7471-7477). ICP0 is thought to be involved in neurovirulence. In cell culture, ICP0 has been found to be required for reactivation from the latent period (Leib et al., 1989, Journal of Virology, 63:759-768). RL2 deletion mutants have been shown to be unable to replicate in vitro (Sacks and Schaffer, 1987, Journal of Virology, 61(3):829-839). In some embodiments, knocking out RL2 renders HSV-1 and / or HSV-2 unable to reactivate from the latent period. In some embodiments, knocking out or knocking down RL2 renders HSV-1 and / or HSV-2 unable to replicate. In some implementations, knocking out or knocking down RL2 can render HSV-1 and / or HSV-2 unable to infect and / or establish latent infection in nerve tissue.

[0259] LAT encodes the only gene expressed by the herpesvirus during the latent period. The latent period is the time it takes for the virus to establish a quiescent infection in host tissues, typically in neural tissues, including the trigeminal ganglion or sacral ganglion. LAT is thought to be involved in the reactivation of herpesvirus infection, allowing the virus to reinfect epithelial and other tissues. In some embodiments, knocking out or knocking down LAT can disable the latent period and / or reactivation of the HSV-1 and / or HSV-2 genes, disrupting the ability of HSV-1 and / or HSV-2 to maintain latent infection and / or reactivation after latent infection. In some embodiments, knocking out or knocking down LAT expression eliminates latent infection of HSV-1 and / or HSV-2. In some embodiments, knocking out or knocking down LAT expression shortens the duration of HSV-1 and / or HSV-2 infection, and treats and / or cures HSV-1 and / or HSV-2 infection.

[0260] RS1 plays a crucial role in the expression of immediate early genes by HSV-1 and HSV-2. RS1 is one of five immediately early genes expressed by herpesviruses and is a major transcriptional regulator. RS1 encodes the viral protein ICP4. ICP4 is important for controlling the overall expression of early and late genes produced by HSV-1 and HSV-2. The RS1 gene is similar in HSV-1 and HSV-2.

[0261] In some embodiments, knocking out or downsampling the RS1, RL2, and / or LAT genes inactivates HSV-1 and / or HSV-2 gene expression, or reduces one or more of viral replication, assembly, maturation, packaging, or infection. In some embodiments, knocking out RS1, RL2, and / or LAT gene expression shortens the duration of HSV-1 and / or HSV-2 infection. In some embodiments, knocking out or downsampling RS1, RL2, and / or LAT gene expression treats or cures HSV-1 and / or HSV-2 infection.

[0262] In some implementations, reducing the duration, amount, and / or frequency of eye-related HSV reactivation can reduce the risk of permanent visual impairment in subjects infected with HSV-1 and / or HSV-2.

[0263] In some embodiments, knocking out and / or knocking down the RS1, RL2, and / or LAT genes, alone or in combination, can make HSV-1 and / or HSV-2 more susceptible to antiviral therapy. Mutations in important genes can make HSV-1, HSV-2, and other viruses more susceptible to antiviral treatment (Zhou et al., Journal of Virology, 2014; 88(19):11121-11129). Knocking out or knocking down the RL2 and LAT and / or RS1 genes, alone or in combination, can be combined with antiviral therapies to treat, prevent, and / or reduce HSV-1 and / or HSV-2 infection. The compositions and methods described in this invention can be used in combination with another antiviral treatment (e.g., another anti-HSV-1 treatment or anti-HSV-2 treatment) to treat, prevent, and / or reduce HSV-1 or HSV-2 infection.

[0264] In one method, one, two, or three RS1, RL2, and LAT genes are targeted for knockout or knockdown, for example, to inhibit one or more viral functions, including, for example, viral gene regulation, viral gene transcription, viral genome replication, expression of latent viral genes, and viral capsid formation. In some embodiments, the method includes knocking out one HSV-1 and HSV-2 gene (e.g., RS1, RL2, or LAT). In some embodiments, the method includes knocking down one HSV-1 and HSV-2 gene (e.g., RS1, RL2, or LAT). In some embodiments, the method includes knocking out two HSV-1 and / or HSV-2 genes, for example, RL2 and LAT genes, for example, RL2 and RS1 genes, for example, RS1 and LAT genes. In some embodiments, the method includes knocking down two HSV-1 and / or HSV-2 genes, for example, RL2 and LAT genes, for example, RL2 and RS1 genes, for example, RS1 and LAT genes. In some embodiments, the method includes knocking out three HSV-1 and / or HSV-2 genes, for example, all three RL2, LAT, and RS1 genes. In some embodiments, the method includes knocking down three HSV-1 and / or HSV-2 genes, for example, all three RS1, RL2, and LAT genes.

[0265] In some implementations, inhibiting one or more viral functions (e.g., viral gene regulation, viral gene transcription, viral genome replication, and viral capsid formation) reduces the duration of primary or recurrent infection and / or reduces viral particle shedding. Subjects may also experience shorter duration of illness, reduced risk of transmission to sexual partners, reduced risk of transmission to the fetus in the event of pregnancy, and / or the potential for complete clearance of HSV-1 and / or HSV-2 (cure).

[0266] Knockout or knockdown of one or more copies of one or more target genes (e.g., RS1, RL2, or LAT genes) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more copies) may be performed at or after the onset of disease, preferably in the early stages of the disease process.

[0267] In some implementations, the method includes initiating treatment on the subject before the onset of the disease.

[0268] In some implementations, the method includes initiating treatment of the subject after the onset of the disease.

[0269] In some embodiments, the method includes initiating treatment on the subject after the onset of disease, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 24, 36, 48 or more months after the onset of HSV-1 and / or HSV-2 infection. In some embodiments, the method includes initiating treatment on the subject after the onset of disease, for example, 1, 2, 3, 4, 5, 10, 15, 20, 25, 40, 50 or 60 years after the onset of HSV-1 and / or HSV-2 infection.

[0270] In some embodiments, the method includes initiating treatment on the subject at a late stage of the disease, such as during the acute or latent phase. In some embodiments, the method includes initiating treatment on the subject during a severe acute phase of the disease affecting the central nervous system, eyes, oropharynx, genital region, and / or other areas.

[0271] Overall, it is expected that starting treatment at all stages of the disease will improve healing, reduce the duration of the disease, and benefit the subjects.

[0272] In some embodiments, the method includes initiating treatment of the subject before disease progression. In some embodiments, the method includes initiating treatment of the subject at an early stage of the disease, for example, when the subject has been exposed to HSV-1 and / or HSV-2 or is believed to have been exposed to HSV-1 and / or HSV-2.

[0273] In some embodiments, the method includes initiating treatment of the subject before disease progression. In some embodiments, the method includes initiating treatment of the subject at an early stage of the disease, for example, when the subject is positive for HSV-1 and / or HSV-2 infection but has no signs or symptoms.

[0274] In some implementations, the method includes initiating treatment upon the presence of one or more of the following findings consistent with or related to HSV-1 and / or HSV-2 infection: fever, headache, body aches, anogenital blisters, oral ulcers, encephalitis, or keratitis.

[0275] In some embodiments, the method includes initiating treatment of the subject when painful blistering occurs in or around the oral cavity (e.g., in the mouth or oropharynx, such as in an infant, child, adult, or young adult).

[0276] In some implementations, the method includes initiating treatment on a subject when, for example, an infant, child, adult, or young adult presents with painful blisters, genital ulcers, and / or flu-like symptoms in the anorectal area.

[0277] In some embodiments, the method includes initiating treatment on a subject suspected of having HSV-1 and / or HSV-2 meningitis and / or HSV-1 and / or HSV-2 encephalitis.

[0278] In some implementations, the method includes initiating treatment upon the presence of one or more of the following symptoms consistent with or related to HSV-1 and / or HSV-2 meningitis and / or encephalitis: fever, headache, vomiting, photophobia, seizures, decreased level of consciousness, lethargy, or somnolence.

[0279] In some embodiments, the method includes initiating treatment upon the presence of any of the following signs consistent with or related to HSV-1 and / or HSV-2 meningitis and / or encephalitis: positive CSF cultures for HSV-1 and / or HSV-2, elevated WBC counts in CSF, and signs of neck stiffness / positive Bruzinski's sign. In some embodiments, the method includes initiating treatment in patients with HSV-1 and / or HSV-2 encephalitis and / or meningitis consistent with EEG, CSF examination, MRI, PCR of CSF specimens, and / or PCR of brain biopsy specimens.

[0280] In some implementations, the method includes initiating treatment upon the presence of any of the following symptoms consistent with or related to visual HSV-1 and / or HSV-2: pain, photophobia, blurred vision, tearing, redness / injection, vision loss, floaters, or flashes of light.

[0281] In some embodiments, the method includes initiating treatment upon the presence of any of the following findings on an ophthalmological examination consistent with or related to HSV-1 and / or HSV-2 (also known as HSV-1 and / or HSV-2 keratitis): small, protruding, clear vesicles on the corneal epithelium; irregular, punctate epithelial erosions on the corneal surface; dense stromal infiltration; ulceration; necrosis; focal, multifocal, or diffuse cellular infiltration; immune rings; neovascularization; or shadow vessels of any grade on the cornea.

[0282] In some embodiments, the method includes initiating treatment upon the presence of any of the following findings on an ophthalmological examination consistent with or related to HSV-1 and / or HSV-2 retinitis or acute retinal necrosis: decreased visual acuity; uveitis; vitreitis; scleral injection; inflammation of the anterior chamber and / or vitreous cavity; vitreous opacity; optic nerve edema; peripheral retinal leukopenia; retinal tear; retinal detachment; retinal necrosis; occlusive vascular disease with evidence of arterial involvement, including arteriole sheath and arteriole attenuation.

[0283] In some embodiments, the method includes initiating treatment upon the appearance of symptoms and / or signs consistent with or related to HSV-1 or HSV-2 infection in the eyes, oropharynx, anorectal region, or central nervous system. In some embodiments, it is advantageous to initiate treatment for suspected HSV-1 or HSV-2 infection early in the course of the disease.

[0284] In some embodiments, the method includes initiating intrauterine treatment. In some embodiments, the subject is at high risk of mother-to-child transmission.

[0285] In some embodiments, the method includes initiating treatment during pregnancy if the mother has an active HSV-1 and / or HSV-2 infection or has a recent primary HSV-1 and / or HSV-2 infection.

[0286] In some implementations, the method includes initiating treatment before or immediately after organ transplantation.

[0287] In some implementations, the method includes initiating treatment in the event of suspected exposure to HSV-1 and / or HSV-2.

[0288] In some implementations, the method includes initiating treatment prophylactically in cases of suspected HSV encephalitis or meningitis.

[0289] In some implementations, HIV-positive subjects and post-transplant subjects are considered to be at risk of severe HSV-1 and / or HSV-2 activation or reactivation due to immunodeficiency, including HSV-encephalitis and meningitis. Newborns are also at risk of severe HSV-encephalitis during delivery due to mother-to-child transmission. Suppressing one or more viral functions (e.g., viral gene regulation, viral gene transcription, viral genome replication, and viral capsid formation) could provide excellent protection for said populations at risk of severe HSV-1 and / or HSV-2 infection. Subjects may experience a low incidence of HSV-1 and / or HSV-2 encephalitis and / or a low incidence of severe neurological sequelae following HSV-1 and / or HSV-2 encephalitis, which would significantly improve their quality of life.

[0290] In some implementations, the method includes initiating treatment on subjects who have or are at risk of developing severe manifestations of HSV-1 and / or HSV-2 infection, such as newborns, subjects with HIV, subjects undergoing immunosuppressive therapy (e.g., after organ transplantation), subjects with cancer, subjects receiving chemotherapy, subjects who will receive chemotherapy, subjects receiving radiation therapy, and subjects who will receive radiation therapy.

[0291] In some implementations, HIV-positive and post-transplant subjects may experience severe HSV-1 and / or HSV-2 activation or reactivation due to immunodeficiency, including HSV-encephalitis and meningitis. Newborns are also at risk of severe HSV-encephalitis during delivery due to mother-to-child transmission. Suppressing key viral functions (e.g., viral gene regulation, viral gene transcription, expression of latent viral genes, viral genome replication, and viral capsid formation) can provide excellent protection for these populations at risk of severe HSV-1 and / or HSV-2 infection. Subjects may experience a low incidence of HSV-1 and / or HSV-2 encephalitis and / or a low incidence of severe neurological sequelae following HSV-1 and / or HSV-2 encephalitis, which will significantly improve their quality of life.

[0292] In some implementations, the method includes initiating treatment on subjects who test positive for HSV-1 and / or HSV-2.

[0293] In some embodiments, the method includes initiating treatment on subjects who test positive for HSV-1 and / or HSV-2 infection. For example, HSV-1 and / or HSV-2 infection can be tested using viral culture, direct fluorescent antibody studies, skin biopsy, PCR, blood serological testing, CSF serological testing, CSF PCR, or brain biopsy. In some embodiments, the method includes initiating treatment on subjects who test positive for HSV-2 infection via diagnostic vitrectomy, intraretinal biopsy, or aqueous fluid PCR, or PCR on vitreous samples.

[0294] In some implementations, the method includes initiating treatment in subjects exposed to HSV-1 and / or HSV-2 and suffering from severe sequelae of high-risk HSV infection.

[0295] In some embodiments, cells are manipulated by editing (e.g., introducing mutations) one or more target genes, such as RS1, RL2, or LAT genes. In some embodiments, for example, the expression of one or more target genes (e.g., one or more RS1, RL2, or LAT genes as described in this invention) is regulated in vivo.

[0296] In some embodiments, the method includes delivering gRNA molecules via adeno-associated virus (AAV). In some embodiments, the method includes delivering gRNA molecules via lentivirus (LV). In some embodiments, the method includes delivering gRNA molecules via nanoparticles.

[0297] In some embodiments, the method further includes administering a second antiviral therapy or treatment agent to the subject, such as the anti-HSV-1 or anti-HSV-2 therapy or treatment agent described in this invention. The composition and other treatments or treatment agents may be administered in any order. For example, the composition described in this invention may be administered simultaneously, before, or after one or more other treatment agents or treatment agents. In some embodiments, the effects of two or more therapies or treatment agents are synergistic. Exemplary anti-HSV-1 and anti-HSV-2 therapies and treatment agents include, but are not limited to, acyclovir, valacyclovir, famciclovir, penciclovir, or vaccines.

[0298] 4. Methods for altering the RS1, RL2, and / or LAT genes

[0299] The RS1, RL2, and / or LAT genes disclosed in this invention can be altered using the genome editing systems, compositions, and methods described in this invention.

[0300] The present invention provides the methods, genome editing systems, and compositions for altering (e.g., knocking out or knocking down) HSV RS1 targets, HSV RL2 targets, or HSV LAT targets.

[0301] The HSV RS1, HSV RL2, or HSV LAT targets disclosed in this invention can be altered individually or in combination through gene editing, for example, using the CRISPR-Cas9-mediated methods, genome editing systems, and compositions described in this invention. Alterations to the HSV RS1, HSV RL2, or HSV LAT targets can be achieved, for example, by knocking out or knocking down them:

[0302] (1) Knock out the RS1, RL2, or LAT genes:

[0303] (a) Insertion or deletion of one or more nucleotides immediately adjacent to or within the early coding region of the RS1, RL2, or LAT gene (e.g., NHEJ-mediated insertion or deletion); or

[0304] (b) The deletion includes at least a portion of the genomic sequence or multiple genomic sequences of the RS1, RL2 or LAT gene (e.g., NHEJ-mediated deletion);

[0305] (2) Knockdown of the RS1, RL2 or LAT genes mediated by eiCas9 molecules or eiCas9-fusion proteins by targeting the non-coding regions (e.g., promoter regions) of the RS1, RL2 or LAT genes.

[0306] All methods induce alterations in the RS1, RL2, and / or LAT genes (e.g., knockout or knockdown). Exemplary mechanisms that may be associated with alterations in one or both of the RS1, RL2, and / or LAT genes include, but are not limited to, non-homologous end joining (e.g., classical or alternative), microhomology-mediated end joining (MMEJ), homology-directed repair (e.g., endogenous donor template-mediated), SDSA (synthesis-dependent strand annealing), single-strand annealing, or single-strand intrusion.

[0307] In some embodiments, the methods, genome editing systems, and compositions of the present invention introduce one or more breaks near the early coding regions of the RS1, RL2, and / or LAT genes. In some embodiments, the methods, genome editing systems, and compositions of the present invention introduce two or more breaks lateral to at least a portion of the RS1, RL2, and / or LAT genes. The removal (e.g., deletion) of the two or more breaks comprises at least a portion of the genomic sequence of the RS1, RL2, and / or LAT genes. In some embodiments, the methods of the present invention include knocking down the RS1, RL2, and / or LAT genes mediated by eiCas9 molecules or eiCas9-fusion proteins by targeting promoter regions at HSV RL2 and / or HSV LAT and / or RS1 target knockdown sites. All methods result in alterations (e.g., knockout or knockdown) of the RS1, RL2, and / or LAT genes.

[0308] 4.1 Knockout of RS1, RL2, or LAT genes by introducing or deleting indels in the RS1, RL2, or LAT genes

[0309] In some embodiments, the method includes introducing an insertion or deletion of one or more nucleotides at an HSV RS1 target knockout site, an HSV RL2 target knockout site, or an HSV LAT target knockout site (e.g., an early coding region) immediately adjacent to the RS1, RL2, and / or LAT gene. As described in this invention, in some embodiments, the method includes introducing one or more breaks (e.g., single-strand or double-strand breaks) in an early coding region (e.g., the 5' or 3' end) sufficiently close to the HSV RL2 target knockout site or the HSV LAT target knockout site, thereby reasonably anticipating that the break-induced indel crosses the HSV RL2 target knockout site or the HSV LAT target knockout site (e.g., an early coding region). NHEJ-mediated break repair allows the introduction of NHEJ-mediated indels in early coding regions adjacent to or at the HSV RL2 target knockout site or the HSV LAT target knockout site.

[0310] In some embodiments, the method includes introducing a deletion of at least a portion of the genomic sequence comprising the RS1, RL2, and / or LAT genes. As described in this invention, in one embodiment, the method includes introducing a double-strand break at one 5' or the other 3' (i.e., flanking) of the RL2, LAT, or RS1 target site. In one embodiment, two gRNAs (e.g., single-molecule (or chimeric) or modular gRNA molecules) are configured to position the two double-strand breaks on opposite sides of the RL2, LAT, or RS1 target knockout sites in the RS1, RL2, and / or LAT genes.

[0311] In some embodiments, single-strand breaks are introduced at or near the HSV RS1 target, HSV RL2 target, or HSV LAT target (e.g., localized by a single gRNA molecule). In some embodiments, a single gRNA molecule (e.g., having a Cas9 nickase) is used to generate single-strand breaks at or near the HSV RS1 target, HSV RL2 target, or HSV LAT target, for example, the gRNA molecule is configured to localize the single-strand breaks upstream (e.g., within 200 bp upstream) or downstream (e.g., within 200 bp downstream) of the HSV RS1 target, HSV RL2 target, or HSV LAT target. In some embodiments, the breaks are localized to avoid unwanted target chromosomal elements, such as repetitive elements, e.g., Alu repeats.

[0312] In some embodiments, double-strand breaks are introduced at or near the HSV RS1 target, HSV RL2 target, or HSV LAT target (e.g., localized by a single gRNA molecule). In some embodiments, a single gRNA molecule (e.g., having a Cas9 nuclease instead of a Cas9 nickase) is used to generate double-strand breaks at or near the HSV RS1 target, HSV RL2 target, or HSV LAT target, for example, the gRNA molecule is configured to localize the double-strand breaks upstream (e.g., within 200 bp upstream) or downstream (e.g., within 200 bp downstream) of the HSV RS1 target, HSV RL2 target, or HSV LAT target. In some embodiments, the breaks are localized to avoid the appearance of undesirable target chromosomal elements, such as repetitive elements, e.g., Alu repeats.

[0313] In some embodiments, two single-strand breaks are introduced at or near the HSV RS1 target, HSV RL2 target, or HSV LAT target (e.g., localized by two gRNA molecules). In some embodiments, two gRNA molecules (e.g., having one or two Cas9 nickases) are used to generate two single-strand breaks at or near the HSV RS1 target, HSV RL2 target, or HSV LAT target, for example, the gRNA molecules are configured such that the single-strand breaks are both localized upstream (e.g., within 200 bp upstream) or downstream (e.g., within 200 bp downstream) of the HSV RS1 target, HSV RL2 target, or HSV LAT target. In some embodiments, two gRNA molecules (e.g., having two Cas9 nickases) are used to generate two single-strand breaks at or close to an HSV RS1 target, an HSV RL2 target, or an HSV LAT target. For example, the gRNA molecules are configured to locate one single-strand break upstream (e.g., within 200 bp) of the HSV RS1, HSV RL2, or HSV LAT target, and to locate the other downstream (e.g., within 200 bp). In some embodiments, the breaks are located to avoid unwanted target chromosomal elements, such as repetitive elements, for example, Alu repeats.

[0314] In some embodiments, two double-strand breaks are introduced at or close to the HSV RS1 target, HSV RL2 target, or HSV LAT target (e.g., localized by two gRNA molecules). In some embodiments, two gRNA molecules (e.g., having one or two Cas9 nucleases instead of Cas9 nickases) are used to generate two double-strand breaks flanking the HSV RS1 target, HSV RL2 target, or HSV LAT target. For example, the gRNA molecules are configured to localize one double-strand break upstream (e.g., within 200 bp) of the HSV RS1 target, HSV RL2 target, or HSV LAT target, and to localize the second double-strand break downstream (e.g., within 200 bp). In some embodiments, the breaks are localized to avoid unwanted target chromosomal elements, such as repetitive elements, e.g., Alu repeats.

[0315] In some embodiments, a double-strand break and two single-strand breaks are introduced at or close to the HSV RS1 target, HSV RL2 target, or HSV LAT target (e.g., localized by three gRNA molecules). In some embodiments, three gRNA molecules (e.g., having a Cas9 nuclease instead of a Cas9 nickase and one and two Cas9 nickases) are used to generate a double-strand break and two single-strand breaks flanking the HSV RS1 target, HSV RL2 target, or HSV LAT target, for example, the gRNA molecules are configured to localize the double-strand break upstream or downstream of the HSV RS1 target, HSV RL2 target, or HSV LAT target (e.g., within 200 bp upstream or downstream), and to localize the two single-strand breaks in opposite locations, for example, downstream or upstream of the HSV RS1 target, HSV RL2 target, or HSV LAT target (e.g., within 200 bp downstream or upstream). In some implementations, the break is located to avoid the appearance of undesirable target chromosomal elements, such as repeating elements, for example, Alu repeats.

[0316] In some embodiments, four single-strand breaks (e.g., localized by four gRNA molecules) are introduced at or close to the HSV RS1 target, HSV RL2 target, or HSV LAT target. In some embodiments, four gRNA molecules (e.g., having one or more Cas9 nickases) are used to generate four single-strand breaks flanking the HSV RS1 target, HSV RL2 target, or HSV LAT target. For example, the gRNA molecules are configured such that the first and second single-strand breaks are localized upstream (e.g., within 200 bp) of the HSV RS1 target, HSV RL2 target, or HSV LAT target, and the third and fourth are localized downstream (e.g., within 200 bp) of the HSV RS1 target, HSV RL2 target, or HSV LAT target. In some embodiments, the breaks are localized to avoid unwanted target chromosomal elements, such as repetitive elements, e.g., Alu repeats.

[0317] In some implementations, two or more (e.g., three or four) gRNA molecules are used with one Cas9 molecule or Cas9-fusion protein. In some implementations, when two or more (e.g., three or four) gRNAs are used with two or more Cas9 molecules, at least one Cas9 molecule originates from a different species than the others. For example, when two gRNA molecules are used with two Cas9 molecules, one Cas9 molecule may originate from one species, while the other Cas9 molecule may originate from a different species. If desired, all Cas9 species are used to generate single-strand or double-strand breaks.

[0318] 4.2. Knockout of one or more of the RS1, RL2, and / or LAT genes by deleting at least a portion of the genomic sequence or multiple genomic sequences comprising the RS1, RL2, and / or LAT genes (e.g., NMEJ-mediated deletion).

[0319] In some embodiments, the method includes deleting a genomic sequence comprising at least a portion of the RS1, RL2, and / or LAT genes, or deleting multiple genomic sequences comprising at least a portion of the RS1, RL2, and / or LAT genes (e.g., NHEJ-mediated deletion). In some embodiments, the method includes introducing two double-strand breaks at one 5' or the other 3' (i.e., flanking) of an HSV RS1 target knockout site, an HSV RL2 target knockout site, or an HSV LAT target knockout site. In some embodiments, two gRNAs (e.g., single-molecule (or chimeric) or modular gRNA molecules) are configured to position the two double-strand breaks at opposite positions to the HSV RL2 target knockout site in the RL2 gene. In some embodiments, two gRNAs (e.g., single-molecule (or chimeric) or modular gRNA molecules) are configured to position the two double-strand breaks at opposite positions to the HSV LAT target knockout site in the LAT gene. In some implementations, two gRNAs (e.g., single-molecule (or chimeric) or modular gRNA molecules) are configured to position the two double-strand breaks at opposite locations to the HSV RS1 target knockout sites in the RS1 gene.

[0320] 4.3. Knockdown of one or more of the RS1, RL2, and / or LAT genes mediated by an inactive Cas9 (eiCas9) molecule or eiCas9-fusion protein.

[0321] Targeted knockdown methods reduce or eliminate the expression of functional RS1, RL2, and / or LAT gene products. As described in this invention, in some embodiments, targeted knockdown is mediated by targeting one, two, or three of the RS1, RL2, and / or LAT genes with an enzymatically inactive Cas9 (eiCas9) molecule or an eiCas9-fusion protein (e.g., eiCas9 fused with a transcriptional repressor domain or a chromatin-modifying protein).

[0322] The methods and compositions of this invention can be used to treat or prevent HSV-1 or HSV-2 infection by targeting transcriptional regulatory regions (e.g., promoter regions (e.g., promoter regions controlling transcription of one or more of the RS1, RL2, and / or LAT genes)). In some embodiments, the promoter regions are targeted to knock down the expression of one or more of the RS1, RL2, and / or LAT genes. Targeted knockdown methods reduce or eliminate the expression of functional RS1, RL2, and / or LAT gene products.

[0323] In some embodiments, one or more eiCas9 molecules can be used to block the binding of one or more endogenous transcription factors. In some embodiments, eiCas9 can be fused to chromatin-modifying proteins. Altering chromatin state can lead to decreased expression of target genes. One or more eiCas9 molecules fused to one or more chromatin-modifying proteins can be used to alter chromatin state.

[0324] In some embodiments, eiCas9-mediated reduction in the expression of one or more of the RS1, RL2, and / or LAT genes leads to a reduction and / or cessation of transcription of the RS1, RL2, and / or LAT RNA. In some embodiments, eiCas9-mediated reduction in the expression of one or more of the RS1, RL2, and / or LAT genes leads to a reduction and / or cessation of translation of HSV-1 or HSV-2 proteins (e.g., ICP0 protein and / or LAT protein and / or transcriptional regulator ICP4 protein) encoded by the RS1, RL2, and / or LAT genes.

[0325] In some implementations, eiCas9-mediated reduction in the expression of one or more of the RS1, RL2, and / or LAT genes, alone or in combination, produces any of the following: reduced HSV DNA production, reduced HSV shedding, reduced HSV replication, decreased viral infectivity, reduced viral particle packaging, and reduced production of viral proteins such as ICP0 proteins or transcriptional regulators such as ICP4 proteins.

[0326] In some embodiments, knocking down one or more of the RS1, RL2, and / or LAT genes cures HSV-1 or HSV-2 infection. In some embodiments, knocking down one or more of the RS1, RL2, and / or LAT genes results in functional cure of HSV-1 or HSV-2 infection. In some embodiments, knocking down one or more of the RS1, RL2, and / or LAT genes results in a sustained virological response to HSV-1 or HSV-2 infection. Targeted knockdown during acute exacerbations can reduce viral shedding and replication, leading to reduced inflammation, which can reduce damage to the eyes. In some embodiments, as described in this invention, the eiCas9 molecule can be a Cas9 variant. For example, but not limited to, the Cas9 variant can be a Streptococcus pyogenes Cas9 variant or a Staphylococcus aureus Cas9 variant. In some embodiments, the Streptococcus pyogenes Cas9 variant is an EQR variant. In some embodiments, the Streptococcus pyogenes Cas9 variant is a VRER variant.

[0327] 5. Guide RNA (gRNA) molecules

[0328] As used herein, a gRNA molecule refers to a nucleic acid that promotes the specific targeting or homing of a gRNA / Cas9 molecule complex to a target nucleic acid. A gRNA molecule can be monomolecular (having a single RNA molecule) (e.g., chimeric) or modular (containing more than one, and typically two, separate, RNA molecules). The gRNA molecules provided by this invention include a targeting domain comprising, consisting of, or substantially consisting of a nucleotide sequence that is fully or partially complementary to, constitutes, or is substantially composed of a target domain (also referred to as a “target sequence”). In some embodiments, the gRNA molecule further includes one or more additional domains, including, for example, a first complementary domain, a linker domain, a second complementary domain, a proximal domain, a tail domain, and a 5' extension domain. Each of these domains is discussed in detail below. In some embodiments, one or more domains in the gRNA molecule include nucleotide sequences that are identical to or share sequences homologous with naturally occurring sequences, such as those from Streptococcus pyogenes, Staphylococcus aureus, or Streptococcus thermophilus. In some embodiments, one or more domains in the gRNA molecule include nucleotide sequences that are identical to or share sequence homology with naturally occurring sequences, such as those from Streptococcus pyogenes or Staphylococcus aureus.

[0329] Figure 1A-1I Several exemplary gRNA structures are provided. Regarding the intrastranded or interstranded interactions of the three-dimensional form or activated form of gRNA, highly complementary regions are... Figure 1A-1I It is sometimes shown as a double strand in other depictions provided in this article. Figure 7This describes the nomenclature of the gRNA domain using the gRNA sequence SEQ ID NO:42, which contains a hairpin loop in the tracrRNA-derived region. In some embodiments, the gRNA may contain more than one (e.g., two, three, or more) hairpin loops in this region (see, for example, Figure 1H-1I ).

[0330] In some embodiments, single-molecule or chimeric gRNA is included, preferably from 5' to 3':

[0331] The targeting domain is complementary to a target domain in the RL2, LAT, or RS1 gene, for example, including a targeting domain comprising a nucleotide sequence selected from SEQ ID NO: 208 to 58749;

[0332] First complementary structural domain;

[0333] Connect structural domains;

[0334] The second complementary structural domain (which is complementary to the first complementary structural domain);

[0335] Proximal domain; and

[0336] Optional, tail structure domain.

[0337] In some implementations, the modular gRNA comprises:

[0338] The first chain, which comprises, preferably, from 5' to 3':

[0339] The targeting domain is complementary to a target domain in the RL2, LAT, or RS1 gene, for example, including a targeting domain comprising a nucleotide sequence selected from SEQ ID NO: 208 to 58749;

[0340] The first complementary structural domain; and

[0341] The second chain, which comprises, preferably, from 5' to 3':

[0342] Optionally, the 5' extended structural domain;

[0343] Second complementary structural domain;

[0344] Proximal domain; and

[0345] Optional, tail structure domain.

[0346] 5.1 Targeted structural domain

[0347] The target domain (sometimes alternatively referred to as the guide sequence) comprises, or is substantially composed of, a nucleic acid sequence complementary to or partially complementary to the target nucleic acid sequence in the RL2, LAT, or RS1 gene. The target domain, wholly or partially complementary to the nucleic acid sequence in the RL2, LAT, or RS1 gene that is complementary to or partially complementary to it, is referred to herein as the target domain.

[0348] Methods for selecting target domains are known in the art (see, for example, Fu 2014; Sternberg 2014). Examples of target domains suitable for the methods, compositions, and kits of this invention include nucleotide sequences as described in SEQ ID NO: 208 to 58749.

[0349] The strand of the target nucleic acid containing the target domain is referred to herein as the complementary strand because it is complementary to the target domain sequence. Since the target domain is part of the gRNA molecule, it contains the base uracil (U) rather than thymine (T); conversely, any DNA molecule encoding the gRNA molecule may include thymine instead of uracil. In the target domain / target domain pair, the uracil base in the target domain will pair with the adenine base in the target domain. In some embodiments, the complementarity between the target domain and the target domain is sufficient to allow the Cas9 molecule to target the target nucleic acid.

[0350] In some embodiments, the targeting domain includes a core domain and an optional second domain. In some of these embodiments, the core domain is located at the 3' end of the second domain, and in some of these embodiments, the core domain is located at or near the 3' end of the targeting domain. In some of these embodiments, the core domain consists of about 8 to about 13 nucleotides at the 3' end of the targeting domain, or is substantially composed thereof. In some embodiments, only the core domain is complementary or partially complementary to the corresponding portion of the target domain, and in some of these embodiments, the core domain is completely complementary to the corresponding portion of the target domain. In some embodiments, the second domain is also complementary or partially complementary to a portion of the target domain. In some embodiments, the core domain is complementary or partially complementary to the core domain target within the target domain, while the second domain is complementary or partially complementary to the second target domain within the target domain. In some embodiments, the core domain and the second domain have the same degree of complementarity with their respective corresponding portions of the target structure. In some embodiments, the degree of complementarity between the core domain and its target and the degree of complementarity between the second domain and its target may differ. In some of these implementations, the core domain may have a higher degree of complementarity with its target than the second domain, while in other implementations, the second domain may have a higher degree of complementarity with the core domain.

[0351] In some embodiments, the length of the targeting domain and / or the core domain within the targeting domain is 3 to 100, 5 to 100, 10 to 100, or 20 to 100 nucleotides, and in some of these embodiments, the length of the targeting domain or the core domain is 3 to 15, 3 to 20, 5 to 20, 10 to 20, 15 to 20, 5 to 50, 10 to 50, or 20 to 50 nucleotides. In some embodiments, the length of the targeting domain and / or the core domain within the targeting domain is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides. In some implementations, the length of the targeting domain and / or the core domain within the targeting domain is 6+ / -2, 7+ / -2, 8+ / -2, 9+ / -2, 10+ / -2, 10+ / -4, 10+ / -5, 11+ / -2, 12+ / -2, 13+ / -2, 14+ / -2, 15+ / -2, or 16+ / -2, 20+ / -5, 30+ / -5, 40+ / -5, 50+ / -5, 60+ / -5, 70+ / -5, 80+ / -5, 90+ / -5, or 100+ / -5 nucleotides.

[0352] In some embodiments where the targeting domain includes a core domain, the core domain is 3 to 20 nucleotides in length, and in some of these embodiments, the core domain is 5 to 15 or 8 to 13 nucleotides in length. In some embodiments where the targeting domain includes a second domain, the second domain is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. In some embodiments where the targeting domain comprises a core domain of 8 to 13 nucleotides in length, each of the following: the targeting domain is 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, or 16 nucleotides in length, and the second domain is 13 to 18, 12 to 17, 11 to 16, 10 to 15, 9 to 14, 8 to 13, 7 to 12, 6 to 11, 5 to 10, 4 to 9, or 3 to 8 nucleotides in length.

[0353] In some embodiments, the targeting domain is completely complementary to the target domain. Similarly, in embodiments where the targeting domain comprises a core domain and / or a second domain, one or both of the core and second domains are completely complementary to a corresponding portion of the target domain. In some embodiments, the targeting domain is partially complementary to the target domain, and in some of these embodiments where the targeting domain comprises a core domain and / or a second domain, one or both of the core and second domains are partially complementary to a corresponding portion of the target domain. In some of these embodiments, the targeting domain or the core domain within the targeting domain is at least about 80%, about 85%, about 90%, or about 95% complementary to the target domain or a corresponding portion of the target domain. In some embodiments, the targeting domain and / or the core or second domain within the targeting domain comprises one or more nucleotides that are not complementary to the target domain or a portion thereof, and in some of these embodiments, the targeting domain and / or the core or second domain within the targeting domain comprises 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides that are not complementary to the target domain. In some embodiments, the core domain includes 1, 2, 3, 4, or 5 nucleotides that are not complementary to the corresponding portion of the target domain. In some embodiments where the target domain includes one or more nucleotides that are not complementary to the target domain, one or more of the non-complementary nucleotides are located within five nucleotides at the 5' or 3' end of the target domain. In some of these embodiments, the target domain includes 1, 2, 3, 4, or 5 nucleotides that are not complementary to the target domain within five nucleotides at its 5' end, 3' end, or both 5' and 3' ends. In some embodiments where the target domain includes two or more nucleotides that are not complementary to the target domain, the two or more of the non-complementary nucleotides are adjacent to each other, and in some of these embodiments, the two or more consecutive non-complementary nucleotides are located within five nucleotides at the 5' or 3' end of the target domain. In some embodiments, the two or more consecutive non-complementary nucleotides are both located more than five nucleotides away from the 5' and 3' ends of the target domain.

[0354] In some embodiments, the targeting domain, core domain, and / or second domain do not contain any modifications. In some embodiments, the targeting domain, core domain, and / or second domain, or one or more nucleotides therein, are modified, including but not limited to the modifications described below. In some embodiments, one or more nucleotides of the targeting domain, core domain, and / or second domain may contain 2' modifications (e.g., modifications at the 2' position on the ribose), such as 2-acetylation, such as 2' methylation. In some embodiments, the backbone of the targeting domain may be modified with phosphate thioesters. In some embodiments, modifications to one or more nucleotides of the targeting domain, core domain, and / or second domain make the targeting domain and / or the gRNA containing the targeting domain less susceptible to degradation or more biocompatible, for example, having lower immunogenicity. In some embodiments, the targeting domain and / or the core or second domain includes 1, 2, 3, 4, 5, 6, 7, or 8 or more modifications, and in some of these embodiments, the targeting domain and / or the core or second domain includes 1, 2, 3, or 4 modifications within the five nucleotides at their respective 5' ends, and / or 1, 2, 3, or 4 modifications within the five nucleotides at their respective 3' ends. In some embodiments, the targeting domain and / or the core or second domain includes modifications at two or more consecutive nucleotides.

[0355] In some embodiments where the targeting domain includes a core and a second domain, the core and second domains contain the same number of modifications. In some of these embodiments, neither domain contains any modifications. In other embodiments, the core domain includes more modifications than the second domain, or vice versa. In some embodiments, modifications to one or more nucleotides in the targeting domain (including the core or second domain) are selected to not interfere with targeting efficacy; this can be evaluated by testing candidate modifications using a system described below. gRNAs having candidate targeting domains, which have selected lengths, sequences, complementarity, or modification levels, can be evaluated using a system described below. The candidate targeting domains can be placed alone or with one or more other candidate variations in a gRNA / Cas9 molecule system known to be functional with a selected target and evaluated.

[0356] In some embodiments, all the modified nucleotides are complementary to and capable of hybridizing to the corresponding nucleotides present in the target domain. In some embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 or more modified nucleotides are not complementary to or are unable to hybridize to the corresponding nucleotides present in the target domain.

[0357] 5.2 First and Second Complementary Structural Domains

[0358] The first and second complementary (sometimes alternatively referred to as crRNA-derived hairpin sequences and tracrRNA-derived hairpin sequences, respectively) domains are completely or partially complementary to each other. In some embodiments, the degree of complementarity is sufficient for the two domains to form a double-stranded region under at least some physiological conditions. In some embodiments, the degree of complementarity between the first and second complementary domains, together with other properties of the gRNA, is sufficient to allow the Cas9 molecule to target the target nucleic acid. Examples of the first and second complementary domains are shown in... Figure 1A-1G This was explained in the text.

[0359] In some implementations (see, for example, Figure 1A-1B The first and / or second complementary domains comprise one or more nucleotides that lack complementarity with the corresponding complementary domain. In some embodiments, the first and / or second complementary domains comprise 1, 2, 3, 4, 5, or 6 nucleotides that are not complementary to the corresponding complementary domain. For example, the second complementary domain may contain 1, 2, 3, 4, 5, or 6 nucleotides that are unpaired with the corresponding nucleotide in the first complementary domain. In some embodiments, the nucleotides on the first or second complementary domain that are not complementary to the corresponding complementary domain loop out from the double strand formed between the first and second complementary domains. In some of these embodiments, the unpaired loopout is located on the second complementary domain, and in some of these embodiments, the unpaired region begins at 1, 2, 3, 4, 5, or 6 nucleotides from the 5' end of the second complementary domain.

[0360] In some embodiments, the length of the first complementary domain is 5 to 30, 5 to 25, 7 to 25, 5 to 24, 5 to 23, 7 to 22, 5 to 22, 5 to 21, 5 to 20, 7 to 18, 7 to 15, 9 to 16, or 10 to 14 nucleotides, and in some of these embodiments, the length of the first complementary domain is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the length of the second complementary domain is 5 to 27, 7 to 27, 7 to 25, 5 to 24, 5 to 23, 5 to 22, 5 to 21, 7 to 20, 5 to 20, 7 to 18, 7 to 17, 9 to 16, or 10 to 14 nucleotides, and in some of these embodiments, the length of the second complementary domain is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides. In some embodiments, the lengths of the first and second complementary domains are each independently 6+ / -2, 7+ / -2, 8+ / -2, 9+ / -2, 10+ / -2, 11+ / -2, 12+ / -2, 13+ / -2, 14+ / -2, 15+ / -2, 16+ / -2, 17+ / -2, 18+ / -2, 19+ / -2, or 20+ / -2, 21+ / -2, 22+ / -2, 23+ / -2, or 24+ / -2 nucleotides. In some embodiments, the second complementary domain is longer than the first complementary domain (e.g., by 2, 3, 4, 5, or 6 nucleotides).

[0361] In some embodiments, the first and / or second complementary structural domains each independently comprise three subdomains, which, in the 5' to 3' direction, are: a 5' subdomain, a central subdomain, and a 3' subdomain. In some embodiments, the 5' and 3' subdomains of the first complementary structural domain are fully or partially complementary to the 3' and 5' subdomains of the second complementary structural domain, respectively.

[0362] In some embodiments, the 5' subdomain of the first complementary domain is 4 to 9 nucleotides in length, and in some of these embodiments, the 5' domain is 4, 5, 6, 7, 8, or 9 nucleotides in length. In some embodiments, the 5' subdomain of the second complementary domain is 3 to 25, 4 to 22, 4 to 18, or 4 to 10 nucleotides in length, and in some of these embodiments, the 5' domain is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the central subdomain of the first complementary domain is 1, 2, or 3 nucleotides in length. In some embodiments, the central subdomain of the second complementary domain is 1, 2, 3, 4, or 5 nucleotides in length. In some embodiments, the 3' subdomain of the first complementary domain is 3 to 25, 4 to 22, 4 to 18, or 4 to 10 nucleotides in length, and in some of these embodiments, the 3' subdomain is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the 3' subdomain of the second complementary domain is 4 to 9 nucleotides in length (e.g., 4, 5, 6, 7, 8, or 9 nucleotides).

[0363] The first and / or second complementary domains may share homology with, or be derived from, naturally occurring or referenced first and / or second complementary domains. In some of these embodiments, the first and / or second complementary domains have at least about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, or about 95% homology with, or are approximately 1, 2, 3, 4, 5, or 6 nucleotides similar to, naturally occurring or referenced first and / or ...

[0364] In some embodiments, the first and / or second complementary domains do not contain any modifications. In other embodiments, the first and / or second complementary domains, or one or more nucleotides therein, are modified, including but not limited to the modifications described below. In some embodiments, one or more nucleotides of the first and / or second complementary domains may contain 2' modifications (e.g., modifications at the 2' position on the ribose), such as 2-acetylation, such as 2' methylation. In some embodiments, the backbone of the targeting domain may be modified with phosphate thioesters. In some embodiments, modifications to one or more nucleotides of the first and / or second complementary domains make the first and / or second complementary domains and / or the gRNA containing the first and / or second complementary domains less susceptible to degradation or more biocompatible, such as having lower immunogenicity. In some embodiments, the first and / or second complementary domains each independently include 1, 2, 3, 4, 5, 6, 7, or 8 or more modifications, and in some of these embodiments, the first and / or second complementary domains each independently include 1, 2, 3, or 4 modifications within five nucleotides at their respective 5', 3', or 5' and 3' ends. In some embodiments, the first and / or second complementary domains each independently do not contain modifications within five nucleotides at their respective 5', 3', or 5' and 3' ends. In some embodiments, one or both of the first and second complementary domains include modifications at two or more consecutive nucleotides.

[0365] In some implementations, modifications to one or more nucleotides in the first and / or second complementary domains are selected to avoid interfering with targeting efficacy. This can be evaluated by testing candidate modifications in a system described below. gRNAs having candidate first and second complementary domains can be evaluated in a system described below, where the first and second complementary domains have selected lengths, sequences, degrees of complementarity, or degrees of modification. The candidate complementary domains can be placed alone or with one or more other candidate variations in a gRNA / Cas9 molecule system known to be functional with the selected target and evaluated.

[0366] In some embodiments, the length of the double-stranded region formed by the first and second complementary domains is, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 bp, excluding any looped or unpaired nucleotides.

[0367] In some embodiments, when double-stranded, the first and second complementary domains contain 11 paired nucleotides (see, for example, gRNA of SEQ ID NO:48). In some embodiments, when double-stranded, the first and second complementary domains contain 15 paired nucleotides (see, for example, gRNA of SEQ ID NO:50). In some embodiments, when double-stranded, the first and second complementary domains contain 16 paired nucleotides (see, for example, gRNA of SEQ ID NO:51). In some embodiments, when double-stranded, the first and second complementary domains contain 21 paired nucleotides (see, for example, gRNA of SEQ ID NO:29). In some embodiments, one or more nucleotides are exchanged between the first and second complementary domains to remove the poly-U bundle. For example, nucleotides 23 and 48 or nucleotides 26 and 45 of the gRNA of SEQ ID NO:48 may be exchanged to produce gRNAs of SEQ ID NO:49 or 31, respectively. Similarly, nucleotides 23 and 39 of the gRNA in SEQ ID NO:29 can be exchanged with nucleotides 50 and 68 to produce the gRNA in SEQ ID NO:30.

[0368] 5.3 Connecting structural domains

[0369] The linker domain is positioned between the first and second complementary domains in a single gRNA or chimeric gRNA and serves to link the first and second complementary domains. Figure 1B-1E Examples of connective domains are provided. In some implementations, a portion of the connective domain originates from a crRNA-derived region, and another portion originates from a tracrRNA-derived region.

[0370] In some embodiments, the linker domain covalently links the first and second complementary domains. In some of these embodiments, the linker domain is composed of or contains covalent bonds. In other embodiments, the linker domain non-covalently links the first and second complementary domains. In some embodiments, the linker domain is ten or fewer nucleotides long, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In other embodiments, the linker domain is longer than 10 nucleotides, such as 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more nucleotides. In some embodiments, the length of the connective domain is 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, 2 to 5, 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20, 10 to 15, 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, or 20 to 25 nucleotides. In some implementations, the length of the connective domain is 10+ / -5, 20+ / -5, 20+ / -10, 30+ / -5, 30+ / -10, 40+ / -5, 40+ / -10, 50+ / -5, 50+ / -10, 60+ / -5, 60+ / -10, 70+ / -5, 70+ / -10, 80+ / -5, 80+ / -10, 90+ / -5, 90+ / -10, 100+ / -5, or 100+ / -10 nucleotides.

[0371] In some embodiments, the connective domain shares homology with, or is derived from, a naturally occurring sequence (e.g., a sequence of tracrRNA where the second complementary domain is 5'). In some embodiments, the connective domain shares homology with, the connective domain disclosed herein (e.g., Figure 1B-1E The linker domain has at least about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% homology or is approximately 1, 2, 3, 4, 5, or 6 nucleotides similar to it.

[0372] In some embodiments, the linker domain does not contain any modifications. In other embodiments, the linker domain or one or more nucleotides therein are modified, including but not limited to the modifications described below. In some embodiments, one or more nucleotides of the linker domain may contain 2' modifications (e.g., modifications at the 2' position on the ribose), such as 2-acetylation, such as 2' methylation. In some embodiments, the backbone of the linker domain may be modified with phosphate thioesters. In some embodiments, modifications to one or more nucleotides of the linker domain make the linker domain and / or the gRNA containing the linker domain less susceptible to degradation or more biocompatible, such as having lower immunogenicity. In some embodiments, the linker domain includes 1, 2, 3, 4, 5, 6, 7, or 8 or more modifications, and in some of these embodiments, the linker domain includes 1, 2, 3, or 4 modifications within five nucleotides at its 5' and / or 3' ends. In some embodiments, the linker domain includes modifications at two or more consecutive nucleotides.

[0373] In some implementations, modifications to one or more nucleotides in the linker domain are selected to avoid interfering with targeting efficacy. This can be evaluated by testing candidate modifications in a system described below. gRNAs having candidate linker domains, which have selected lengths, sequences, complementarity, or modification levels, can be evaluated in a system described below. The candidate linker domains can be placed alone or with one or more other candidate variations in a gRNA / Cas9 molecule system known to be functional with the selected target and evaluated.

[0374] In some embodiments, the linker domain comprises a double-stranded region typically adjacent to the 3' end of the first complementary domain and / or the 5' end of the second complementary domain, or within 1, 2, or 3 nucleotides therein. In some of these embodiments, the length of the double-stranded region of the linker domain is 10+ / -5, 15+ / -5, 20+ / -5, 20+ / -10, or 30+ / -5 bp. In some embodiments, the length of the double-stranded region of the linker domain is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 bp. In some embodiments, the sequences forming the double-stranded region of the linker domain are completely complementary. In other embodiments, one or both sequences forming the double-stranded region contain one or more nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides) that are not complementary to the other double-stranded sequences.

[0375] 5.4 5' Extended Structural Domain

[0376] In some implementations, the modular gRNA disclosed herein includes a 5' extension domain, that is, one or more additional nucleotides at the 5' of the second complementary domain (see, for example, Figure 1A In some embodiments, the length of the 5' extension domain is 2 to 10 or more, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, or 2 to 4 nucleotides, and in some of these embodiments, the length of the 5' extension domain is 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides.

[0377] In some embodiments, the 5' extension domain nucleotide does not contain modifications, such as those of the types provided below. However, in some embodiments, the 5' extension domain contains one or more modifications, such as modifications that make it less susceptible to degradation or more biocompatible (e.g., lower immunogenicity). As an example, the backbone of the 5' extension domain can be modified with phosphate thioesters, or one or more other modifications as described below. In some embodiments, the 5' extension domain nucleotide may contain 2' modifications (e.g., modifications at the 2' position on the ribose), such as 2-acetylation, such as 2' methylation, or one or more other modifications as described below.

[0378] In some embodiments, the 5' extension domain may contain up to 1, 2, 3, 4, 5, 6, 7, or 8 modifications. In some embodiments, the 5' extension domain contains up to 1, 2, 3, or 4 modifications within the 5 nucleotides at its 5' end, for example in modular gRNA molecules. In some embodiments, the 5' extension domain contains up to 1, 2, 3, or 4 modifications within the 5 nucleotides at its 3' end, for example in modular gRNA molecules.

[0379] In some embodiments, the 5' extension domain includes modifications at two consecutive nucleotides, such as within the 5' terminus of the 5' extension domain, within the 5' terminus of the 5' extension domain, or within two consecutive nucleotides at one or both ends of the 5' extension domain. In some embodiments, no two consecutive nucleotides are modified within the 5' terminus of the 5' extension domain, within the 5' terminus of the 5' extension domain, or within a region at one or both ends of the 5' extension domain. In some embodiments, no nucleotides are modified within the 5' terminus of the 5' extension domain, within the 5' terminus of the 5' extension domain, or within a region at one or both ends of the 5' extension domain.

[0380] Modifications to the 5' extension domain can be selected to avoid interfering with the efficacy of the gRNA molecule. This can be evaluated by testing candidate modifications in the systems described below. gRNAs with candidate 5' extension domains, having selected lengths, sequences, complementarity, or modification levels, can be evaluated in the systems described below. Candidate 5' extension domains can be placed alone or with one or more other candidate variations in gRNA / Cas9 molecule systems known to be functional with selected targets and evaluated.

[0381] In some embodiments, the 5' extension domain is related to a reference 5' extension domain (e.g., a naturally occurring (e.g., Streptococcus pyogenes, Staphylococcus aureus, or Streptococcus thermophilus) 5' extension domain) or a 5' extension domain described herein (e.g., from...). Figure 1A-1G It has at least about 60%, about 70%, about 80%, about 85%, about 90% or about 95% homology, or is approximately 1, 2, 3, 4, 5 or 6 nucleotides similar to it.

[0382] 5.5 Proximal domain

[0383] Figure 1A-1G An instance of a near-end structure is provided.

[0384] In some embodiments, the proximal domain is 5 to 20 or more nucleotides long, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides long. In some of these embodiments, the proximal domain is 6+ / -2, 7+ / -2, 8+ / -2, 9+ / -2, 10+ / -2, 11+ / -2, 12+ / -2, 13+ / -2, 14+ / -2, 14+ / -2, 16+ / -2, 17+ / -2, 18+ / -2, 19+ / -2, or 20+ / -2 nucleotides long. In some embodiments, the proximal domain is 5 to 20, 7 to 18, 9 to 16, or 10 to 14 nucleotides long.

[0385] In some embodiments, the proximal domain may share homology with or be derived from a naturally occurring proximal domain. In some of these embodiments, the proximal domain is related to proximal domains disclosed herein (e.g., the proximal domains of Streptococcus pyogenes, Staphylococcus aureus, or Streptococcus thermophilus, including…). Figure 1A-1G Those described herein have at least about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, or about 95% homology, or are approximately 1, 2, 3, 4, 5, or 6 nucleotides similar to them.

[0386] In some embodiments, the proximal domain does not contain any modifications. In other embodiments, the proximal domain or one or more nucleotides therein are modified, including but not limited to modifications set forth herein. In some embodiments, one or more nucleotides of the proximal domain may contain 2' modifications (e.g., modifications at the 2' position on the ribose), such as 2-acetylation, such as 2' methylation. In some embodiments, the backbone of the proximal domain may be modified with phosphate thioesters. In some embodiments, modifications to one or more nucleotides of the proximal domain make the proximal domain and / or the gRNA containing the proximal domain less susceptible to degradation or more biocompatible, such as having lower immunogenicity. In some embodiments, the proximal domain includes 1, 2, 3, 4, 5, 6, 7, or 8 or more modifications, and in some of these embodiments, the proximal domain includes 1, 2, 3, or 4 modifications within five nucleotides at its 5' and / or 3' ends. In some embodiments, the proximal domain includes modifications at two or more consecutive nucleotides.

[0387] In some embodiments, modifications to one or more nucleotides in the proximal domain are selected to avoid interfering with targeting efficacy. This can be evaluated by testing candidate modifications in a system described below. gRNAs having candidate proximal domains, which have selected lengths, sequences, complementarity, or modification levels, can be evaluated in a system described below. The candidate proximal domains can be placed alone or with one or more other candidate variations in a gRNA / Cas9 molecule system known to be functional with the selected target and evaluated.

[0388] 5.6 Tail Structural Domain

[0389] The broad-spectrum tail domain is suitable for use in the gRNA molecules disclosed herein. Figure 1A and 1C -1G provides an instance of this tail structure field.

[0390] In some embodiments, the tail domain is absent. In other embodiments, the tail domain is 1 to 100 or more nucleotides in length, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides in length. In some embodiments, the tail domain is 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 50, 10 to 100, 20 to 100, 10 to 90, 20 to 90, 10 to 80, 20 to 80, 10 to 70, 20 to 70, 10 to 60, 20 to 60, 10 to 50, 20 to 50, 10 to 40, 20 to 40, 10 to 30, 20 to 30, 20 to 25, 10 to 20, or 10 to 15 nucleotides in length. In some embodiments, the length of the tail domain is 5+ / -5, 10+ / -5, 20+ / -10, 20+ / -5, 25+ / -10, 30+ / -10, 30+ / -5, 40+ / -10, 40+ / -5, 50+ / -10, 50+ / -5, 60+ / -10, 60+ / -5, 70+ / -10, 70+ / -5, 80+ / -10, 80+ / -5, 90+ / -10, 90+ / -5, 100+ / -10, or 100+ / -5 nucleotides.

[0391] In some embodiments, the tail domain may share homology with, or be derived from, a naturally occurring tail domain or the 5' end of a naturally occurring tail domain. In some of these embodiments, the proximal domain shares homology with naturally occurring tail domains disclosed herein (e.g., tail domains of *Streptococcus pyogenes*, *Staphylococcus aureus*, or *Streptococcus thermophilus*, including…). Figure 1A and Figure 1C-1G Those described herein have at least about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, or about 95% homology, or are approximately 1, 2, 3, 4, 5, or 6 nucleotides similar to them.

[0392] In some embodiments, the tail domain comprises sequences that are complementary to each other and form a double-stranded region under at least some physiological conditions. In some of these embodiments, the tail domain comprises a tail double-stranded domain that can form a tail double-stranded region. In some embodiments, the tail double-stranded region is 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 bp in length. In some embodiments, the tail domain comprises a 3' single-stranded domain that does not form a double-stranded tail double-stranded domain. In some of these embodiments, the single-stranded domain is 3 to 10 nucleotides long (e.g., 3, 4, 5, 6, 7, 8, 9, 10) or 4 to 6 nucleotides long.

[0393] In some embodiments, the tail domain does not contain any modifications. In other embodiments, the tail domain or one or more nucleotides therein are modified, including but not limited to modifications set forth herein. In some embodiments, one or more nucleotides of the tail domain may contain 2' modifications (e.g., modifications at the 2' position on the ribose), such as 2-acetylation, such as 2' methylation. In some embodiments, the backbone of the tail domain may be modified with phosphate thioesters. In some embodiments, modifications to one or more nucleotides of the tail domain make the tail domain and / or the gRNA containing the tail domain less susceptible to degradation or more biocompatible, such as having lower immunogenicity. In some embodiments, the tail domain includes 1, 2, 3, 4, 5, 6, 7, or 8 or more modifications, and in some of these embodiments, the tail domain includes 1, 2, 3, or 4 modifications within five nucleotides at its 5' and / or 3' ends. In some embodiments, the tail domain includes modifications at two or more consecutive nucleotides.

[0394] In some embodiments, the tail domain includes a nucleotide at its 3' end that is associated with an in vitro or in vivo transcription method. When the T7 promoter is used for in vitro transcription of gRNA, these nucleotides can be any nucleotide present before the 3' end of the DNA template. In some embodiments, the gRNA molecule includes a 3' polyadenylated tail prepared by in vitro transcription from a DNA template. In some embodiments, the 5' nucleotide of the target domain of the gRNA molecule is a guanine nucleotide, the DNA template contains a T7 promoter sequence immediately upstream of the sequence corresponding to the target domain, and the 3' nucleotide of the T7 promoter sequence is not a guanine nucleotide. In some embodiments, the 5' nucleotide of the target domain of the gRNA molecule is not a guanine nucleotide, the DNA template contains a T7 promoter sequence immediately upstream of the sequence corresponding to the target domain, and the 3' nucleotide of the T7 promoter sequence is a guanine nucleotide downstream of a nucleotide other than a guanine nucleotide.

[0395] In some embodiments, the tail domain includes a nucleotide at the 3' end that is associated with the in vitro or in vivo transcription method. When the T7 promoter is used for in vitro transcription of gRNA, these nucleotides can be any nucleotide present before the 3' end of the DNA template. When the U6 promoter is used for in vivo transcription, these nucleotides can be the sequence UUUUUU. When the H1 promoter is used for transcription, these nucleotides can be the sequence UUUU. When an alternative pol-III promoter is used, these nucleotides can be a variety of numbers of uracil bases, depending on, for example, the termination signal of the pol-III promoter, or they may include alternative bases.

[0396] In some embodiments, the proximal and tail domains together comprise, or consist substantially of, the sequences shown in SEQ ID NO: 32, 33, 34, 35, 36, or 37.

[0397] 5.7 Exemplary Single / Chimeric gRNA

[0398] In some embodiments, the gRNA of the present invention has the following structure: 5' [targeting domain]-[first complementary domain]-[connector domain]-[second complementary domain]-[proximal domain]-[tail domain]-3', wherein:

[0399] The targeting domain comprises a core domain and optionally a second domain, and is 10 to 50 nucleotides in length;

[0400] The length of the first complementary domain is 5 to 25 nucleotides, and in some embodiments, it has at least about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, or about 95% homology with the reference first complementary domain disclosed herein.

[0401] The length of the connecting domain is 1 to 5 nucleotides;

[0402] The second complementary domain is 5 to 27 nucleotides in length and, in some embodiments, has at least about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, or about 95% homology with the reference second complementary domain disclosed herein.

[0403] The proximal domain is 5 to 20 nucleotides in length and, in some embodiments, has at least about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, or about 95% homology to the reference proximal domain disclosed herein; and

[0404] The tail domain is 1 to 50 nucleotides in length and, in some embodiments, has at least about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, or about 95% homology with the reference tail domain disclosed herein.

[0405] In some embodiments, the sequences from (a), (b), and / or (c) have at least about 50%, about 60%, about 70%, about 75%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 99% homology with the corresponding sequences of naturally occurring gRNAs or with the gRNAs described herein.

[0406] In some embodiments, the single-molecule gRNA as disclosed herein preferably comprises, from 5' to 3':

[0407] Targeting domains, which may contain, for example, 10-50 nucleotides;

[0408] The first complementary structural domain, for example, includes 15, 16, 17, 18, 19, 20, 21, 22, 23.

[0409] 24, 25, or 26 nucleotides;

[0410] Connect structural domains;

[0411] Second complementary structural domain;

[0412] Proximal domain; and

[0413] Tail structural domain,

[0414] in,

[0415] (a) When considered together, the proximal and tail domains contain at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides;

[0416] (b) The 3' end of the last nucleotide of the second complementary domain contains at least 15, 18, 20, 25,

[0417] 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides; or

[0418] (c) The 3' end of the last nucleotide of the second complementary domain contains at least 16, 19, 21, and 26 nucleotides.

[0419] 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides, wherein the nucleotides are complementary to the corresponding nucleotides of the first complementary domain.

[0420] In some embodiments, the sequences from (a), (b), and / or (c) have at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% homology with the corresponding sequences of naturally occurring gRNAs or with the gRNAs described herein.

[0421] In some implementations, when considered together, the proximal and tail domains contain at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0422] In some embodiments, the 3' of the last nucleotide of the second complementary domain has at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0423] In some embodiments, the 3' of the last nucleotide of the second complementary domain has at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides, which are complementary to the corresponding nucleotides of the first complementary domain.

[0424] In some embodiments, the targeting domain comprises, is substantially composed of, or contains thereof 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides that are complementary to or partially complementary to the target domain or thereof, or to a portion thereof. For example, the length of the targeting domain is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides. In some of these embodiments, the targeting domain is complementary to the target domain over its entire length, the entire length of the target domain, or both.

[0425] In some embodiments, the single-molecule or chimeric gRNA molecule disclosed herein (comprising a target domain, a first complementary domain, a linker domain, a second complementary domain, a proximal domain, and optionally a tail domain) comprises the amino acid sequence shown in SEQ ID NO:42, wherein the target domain is listed as 20 N (residues 1-20) but its length can range from 16 to 26 nucleotides, and wherein the last six residues (residues 97-102) represent the termination signal of the U6 promoter, but may be absent or fewer in number. In some embodiments, the single-molecule or chimeric gRNA molecule is a Streptococcus pyogenes gRNA molecule.

[0426] In some embodiments, the single-molecule or chimeric gRNA molecule disclosed herein (comprising a target domain, a first complementary domain, a linker domain, a second complementary domain, a proximal domain, and optionally a tail domain) comprises the amino acid sequence shown in SEQ ID NO:38, wherein the target domain is listed as 20 N (residues 1-20) but its length can range from 16 to 26 nucleotides, and wherein the last six residues (residues 97-102) represent the termination signal of the U6 promoter, but may be absent or fewer in number. In some embodiments, the single-molecule or chimeric gRNA molecule is a Staphylococcus aureus gRNA molecule.

[0427] The sequence and structure of an exemplary chimeric gRNA are also shown in Figure 1H-1I middle.

[0428] 5.8 Exemplary Modular gRNA

[0429] In some implementations, the modular gRNA disclosed herein comprises:

[0430] The first chain, which comprises, preferably, from 5' to 3':

[0431] Targeted structural domains, such as those containing 15, 16, 17, 18, 19, 20, 21, 22, 23,

[0432] 24, 25, or 26 nucleotides;

[0433] The first complementary structural domain; and

[0434] The second chain, which comprises, preferably, from 5' to 3':

[0435] Optionally, the 5' extended structural domain;

[0436] Second complementary structural domain;

[0437] Proximal domain; and

[0438] Tail structural domain,

[0439] in:

[0440] (a) When considered together, the proximal and tail domains contain at least 15, 18, 20, and 25 cells.

[0441] 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides;

[0442] (b) The 3' end of the last nucleotide of the second complementary domain contains at least 15, 18, 20, 25,

[0443] 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides; or

[0444] (c) The 3' end of the last nucleotide of the second complementary domain contains at least 16, 19, 21, and 26 nucleotides.

[0445] 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides, wherein the nucleotides are complementary to the corresponding nucleotides of the first complementary domain.

[0446] In some embodiments, the sequences from (a), (b), or (c) have at least about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 99% homology with the corresponding sequences of naturally occurring gRNAs or with the gRNAs described herein.

[0447] In some implementations, when considered together, the proximal and tail domains contain at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0448] In some embodiments, the 3' end of the last nucleotide of the second complementary domain contains at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides. In some embodiments, the 3' end of the last nucleotide of the second complementary domain contains at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides, said nucleotides being complementary to the corresponding nucleotide of the first complementary domain.

[0449] In some embodiments, the target domain comprises, has, or is composed of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides that are complementary to the target domain (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides), for example, the length of the target domain is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides.

[0450] In some embodiments, the targeting domain comprises, substantially comprises, or contains 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides complementary to the target domain or a portion thereof. In some of these embodiments, the targeting domain is complementary to the target domain over its entire length, the entire length of the target domain, or both.

[0451] In some embodiments, the target domain includes, has, or consists of 16 nucleotides having complementarity with the target domain (e.g., 16 consecutive nucleotides), for example, the target domain is 16 nucleotides in length; when the proximal and tail domains are considered together, they include 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0452] In some embodiments, the target domain includes, has, or is composed of 16 nucleotides having complementarity with the target domain (e.g., 16 consecutive nucleotides), for example, the target domain is 16 nucleotides in length; and the 3' of the last nucleotide of the second complementary domain has at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0453] In some embodiments, the target domain includes, has, or is composed of 16 nucleotides having complementarity with the target domain (e.g., 16 consecutive nucleotides), for example, the target domain is 16 nucleotides long; and the 3' of the last nucleotide in the second complementary domain, which is complementary to the corresponding nucleotide in the first complementary domain, has at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides.

[0454] In some implementations, the targeting domain has or consists of 17 nucleotides having complementarity with the target domain (e.g., 17 consecutive nucleotides), for example, the targeting domain is 17 nucleotides in length; when the proximal and tail domains are considered together, they include 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0455] In some embodiments, the target domain has or is composed of 17 nucleotides that are complementary to the target domain (e.g., 17 consecutive nucleotides), for example, the target domain is 17 nucleotides long; and the 3' of the last nucleotide of the second complementary domain has at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50 or 53 nucleotides.

[0456] In some embodiments, the target domain has or is composed of 17 nucleotides that are complementary to the target domain (e.g., 17 consecutive nucleotides), for example, the target domain is 17 nucleotides long; and the 3' of the last nucleotide in the second complementary domain, which is complementary to the corresponding nucleotide in the first complementary domain, has at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides.

[0457] In some implementations, the targeting domain has or consists of 18 nucleotides having complementarity with the target domain (e.g., 18 consecutive nucleotides), for example, the targeting domain is 18 nucleotides in length; when the proximal and tail domains are considered together, they include 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0458] In some embodiments, the target domain has or is composed of 18 nucleotides having complementarity with the target domain (e.g., 18 consecutive nucleotides), for example, the target domain is 18 nucleotides long; and the 3' of the last nucleotide of the second complementary domain has at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50 or 53 nucleotides.

[0459] In some embodiments, the target domain has or is composed of 18 nucleotides (e.g., 18 consecutive nucleotides) that are complementary to the target domain, for example, the target domain is 18 nucleotides long; and the 3' of the last nucleotide in the second complementary domain, which is complementary to the corresponding nucleotide in the first complementary domain, has at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides.

[0460] In some embodiments, the target domain includes, has, or consists of 19 nucleotides having complementarity with the target domain (e.g., 19 consecutive nucleotides), for example, the target domain is 19 nucleotides in length; when the proximal and tail domains are considered together, they include 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0461] In some embodiments, the target domain includes, has, or is composed of 19 nucleotides having complementarity with the target domain (e.g., 19 consecutive nucleotides), for example, the target domain is 19 nucleotides in length; and the 3' of the last nucleotide of the second complementary domain has at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0462] In some embodiments, the target domain includes, has, or is composed of 19 nucleotides having complementarity with the target domain (e.g., 19 consecutive nucleotides), for example, the target domain is 19 nucleotides long; and the 3' of the last nucleotide in the second complementary domain, which is complementary to the corresponding nucleotide in the first complementary domain, has at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides.

[0463] In some embodiments, the target domain includes, has, or consists of 20 nucleotides having complementarity with the target domain (e.g., 20 consecutive nucleotides), for example, the target domain is 20 nucleotides in length; when the proximal and tail domains are considered together, they include 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0464] In some embodiments, the target domain includes, has, or is composed of 20 nucleotides having complementarity with the target domain (e.g., 20 consecutive nucleotides), for example, the target domain is 20 nucleotides in length; and the 3' of the last nucleotide of the second complementary domain has at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0465] In some embodiments, the target domain includes, has, or is composed of 20 nucleotides having complementarity with the target domain (e.g., 20 consecutive nucleotides), for example, the target domain is 20 nucleotides in length; and the 3' of the last nucleotide in the second complementary domain, which is complementary to the corresponding nucleotide in the first complementary domain, has at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides.

[0466] In some embodiments, the target domain includes, has, or consists of 21 nucleotides having complementarity with the target domain (e.g., 21 consecutive nucleotides), for example, the target domain is 21 nucleotides in length; when the proximal and tail domains are considered together, they include 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0467] In some embodiments, the target domain includes, has, or is composed of 21 nucleotides (e.g., 21 consecutive nucleotides) that are complementary to the target domain, for example, the target domain is 21 nucleotides long; and the 3' of the last nucleotide of the second complementary domain has at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0468] In some embodiments, the target domain includes, has, or is composed of 21 nucleotides having complementarity with the target domain (e.g., 21 consecutive nucleotides), for example, the target domain is 21 nucleotides long; and the 3' of the last nucleotide in the second complementary domain, which is complementary to the corresponding nucleotide in the first complementary domain, has at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides.

[0469] In some embodiments, the target domain includes, has, or consists of 22 nucleotides having complementarity with the target domain (e.g., 22 consecutive nucleotides), for example, the target domain is 22 nucleotides in length; when the proximal and tail domains are considered together, they include 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0470] In some embodiments, the target domain includes, has, or is composed of 22 nucleotides having complementarity with the target domain (e.g., 22 consecutive nucleotides), for example, the target domain is 22 nucleotides long; and the 3' of the last nucleotide of the second complementary domain has at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0471] In some embodiments, the target domain includes, has, or is composed of 22 nucleotides (e.g., 22 consecutive nucleotides) that are complementary to the target domain, for example, the target domain is 22 nucleotides long; and the 3' of the last nucleotide in the second complementary domain, which is complementary to the corresponding nucleotide in the first complementary domain, has at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides.

[0472] In some embodiments, the target domain includes, has, or consists of 23 nucleotides having complementarity with the target domain (e.g., 23 consecutive nucleotides), for example, the target domain is 23 nucleotides in length; when the proximal and tail domains are considered together, they include 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0473] In some embodiments, the target domain includes, has, or is composed of 23 nucleotides having complementarity with the target domain (e.g., 23 consecutive nucleotides), for example, the target domain is 23 nucleotides in length; and the 3' of the last nucleotide of the second complementary domain has at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0474] In some embodiments, the target domain includes, has, or is composed of 23 nucleotides having complementarity with the target domain (e.g., 23 consecutive nucleotides), for example, the target domain is 23 nucleotides in length; and the 3' of the last nucleotide in the second complementary domain, which is complementary to the corresponding nucleotide in the first complementary domain, has at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides.

[0475] In some embodiments, the target domain includes, has, or consists of 24 nucleotides having complementarity with the target domain (e.g., 24 consecutive nucleotides), for example, the target domain is 24 nucleotides in length; when the proximal and tail domains are considered together, they include 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0476] In some embodiments, the target domain includes, has, or is composed of 24 nucleotides having complementarity with the target domain (e.g., 24 consecutive nucleotides), for example, the target domain is 24 nucleotides in length; and the 3' of the last nucleotide of the second complementary domain has at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0477] In some embodiments, the target domain includes, has, or is composed of 24 nucleotides having complementarity with the target domain (e.g., 24 consecutive nucleotides), for example, the target domain is 24 nucleotides long; and the 3' of the last nucleotide in the second complementary domain, which is complementary to the corresponding nucleotide in the first complementary domain, has at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides.

[0478] In some embodiments, the target domain includes, has, or consists of 25 nucleotides having complementarity with the target domain (e.g., 25 consecutive nucleotides), for example, the target domain is 25 nucleotides in length; when the proximal and tail domains are considered together, they include 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0479] In some embodiments, the target domain includes, has, or is composed of 25 nucleotides having complementarity with the target domain (e.g., 25 consecutive nucleotides), for example, the target domain is 25 nucleotides in length; and the 3' of the last nucleotide of the second complementary domain has at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0480] In some embodiments, the target domain includes, has, or is composed of 25 nucleotides having complementarity with the target domain (e.g., 25 consecutive nucleotides), for example, the target domain is 25 nucleotides in length; and the 3' of the last nucleotide in the second complementary domain, which is complementary to the corresponding nucleotide in the first complementary domain, has at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides.

[0481] In some embodiments, the target domain includes, has, or consists of 26 nucleotides having complementarity with the target domain (e.g., 26 consecutive nucleotides), for example, the target domain is 26 nucleotides in length; when the proximal and tail domains are considered together, they include 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0482] In some embodiments, the target domain includes, has, or is composed of 26 nucleotides having complementarity with the target domain (e.g., 26 consecutive nucleotides), for example, the target domain is 26 nucleotides long; and the 3' of the last nucleotide of the second complementary domain has at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0483] In some embodiments, the target domain includes, has, or is composed of 26 nucleotides having complementarity with the target domain (e.g., 26 consecutive nucleotides), for example, the target domain is 26 nucleotides long; and the 3' of the last nucleotide in the second complementary domain, which is complementary to the corresponding nucleotide in the first complementary domain, has at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides.

[0484] 5.9gRNA delivery

[0485] In some embodiments of the method provided by this invention, the method includes the delivery of one or more (e.g., two, three, or four) gRNA molecules as described in this invention. In some of these embodiments, the gRNA molecules are delivered by intravenous injection, intramuscular injection, subcutaneous injection, or inhalation. In some embodiments, the gRNA molecules are delivered together with Cas9 molecules in a genome editing system.

[0486] 6. Methods for designing gRNA

[0487] Methods are provided for selecting, designing, and validating target domains used in the gRNAs described in this invention. The invention also provides exemplary target domains for incorporation into gRNAs.

[0488] Methods for target sequence selection and validation, as well as off-target analysis, have been previously described (see, for example, Mali 2013; Hsu 2013; Fu 2014; Heigwer 2014; Bae 2014; Xiao 2014). For example, software tools can be used to optimize the selection of potential target domains corresponding to a user's target sequence, e.g., to minimize total off-target activity across the genome. Off-target activity can be different from cleavage. For each possible target domain selection using *Streptococcus pyogenes* Cas9, the tool can identify all off-target sequences (above NAG or NGG PAM) across the genome containing up to a certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) mismatched base pairs. Cleavage efficiency at each off-target sequence is predictable, e.g., using an experimentally derived weighting scheme. Each possible target domain is then ranked according to its total predicted off-target cleavage; the highest-ranked target domains represent those likely to have the largest mid-target cleavage and the fewest off-target cleavages. Other functionalities (e.g., automated reagent design for CRISPR construction, primer design for mid-target surveyor assays, and primer design for high-throughput detection and quantification of off-target cleavage via next-generation sequencing) may also be included in the tool. Functional evaluation of candidate target domains and gRNAs containing those target domains can be performed using methods known in the art and / or described herein.

[0489] As a non-limiting example, DNA sequence retrieval algorithms were used to identify the target domains used in gRNAs for use with Cas9 of *Streptococcus pyogenes*, *Staphylococcus aureus*, and *Neisseria meningitidis*. 17-mer and 20-mer target domains were designed for the *Streptococcus pyogenes* and *Neisseria meningitidis* targets, while 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, and 24-mer target domains were designed for the *Staphylococcus aureus* target. gRNA design was performed using custom gRNA design software based on the public tool cas-offinder (Bae 2014). This software scores guides after calculating their genome-wide off-target propensity. Typically, for guides ranging in length from 17 to 24, a range from perfect matches to matches with 7 mismatches was considered. Once off-target sites were determined, a total score for each guide was calculated, and a summary was output in a table using a web interface. In addition to identifying potential target sites adjacent to PAM sequences, the software also identifies all PAM-near sequences that differ from selected target sites by 1, 2, 3, or more than 3 nucleotides. The genomic DNA sequence of each gene is obtained from the UCSC Genome Browser, and the sequences are screened for repetitive elements using the publicly available RepeatMasker program. RepeatMasker retrieves repetitive elements and low-complexity regions from the input DNA sequence. The output is a detailed annotation of the repetitions present in the given query sequence.

[0490] After identification, the target domains were classified according to their distance from the target site, their orthogonality, and the presence of 5'G (based on the identification of close matches in the human genome including the relevant PAM, e.g., NGGPAM for wild-type Streptococcus pyogenes Cas9 molecule); NNGRRT (SEQ ID NO: 204) or NNGRRV (SEQ ID NO: 205) PAM for wild-type Staphylococcus aureus Cas9 molecule, or NNNNGATT or NNNNGCTTPAM for wild-type Neisseria meningitidis Cas9 molecule; PAMs selected from the group consisting of NGAG, NGCG, NGGG, NGTG, NGAA, NGAT, and NGAC for Streptococcus pyogenes Cas9 EQR variants; or PAMs selected from the group consisting of NGCG, NGCA, NGCT, and NGCC for Streptococcus pyogenes Cas9 VRER variants. Orthogonality refers to the number of sequences in the human genome containing the minimum number of mismatches with the target sequence. "High level of orthogonality" or "good orthogonality" can refer, for example, to a 20-mer targeting domain that, apart from the intended target, has neither a consistent sequence in the human genome nor any sequence containing one or both mismatches of the target sequence. Targeting domains with good orthogonality are selected to minimize off-target DNA cleavage.

[0491] Both single-gRNA nuclease cleavage and pairwise "nickase" strategies targeting dual-gRNAs were used to identify target domains. The criteria for selecting target domains and determining which target domains can be used for pairwise "nickase" strategies are based on two considerations:

[0492] (1) The targeting domain should be oriented on the DNA such that the PAM faces outwards, and cleavage with the D10A Cas9 cleavage enzyme will produce a 5' overhang; and

[0493] (2) It is assumed that cleavage with a double-cutting enzyme pair will result in the deletion of the entire insertion sequence at a reasonable frequency. However, cleavage with a double-cutting enzyme pair may also result in an indel mutation at only one site of the gRNA.

[0494] Candidate pairs can be tested on how effectively they remove the entire sequence contrast that causes an indel mutation at the target site of a target domain.

[0495] 6.1 Targeting domain for knocking out the HSV-1 RS1 gene

[0496] The target domains of gRNA used to knock out the HSV-1 RS1 gene in conjunction with the method disclosed in this invention were identified and graded as grade 5 for Streptococcus pyogenes, grade 7 for Staphylococcus aureus, and grade 5 for Neisseria meningitidis.

[0497] For *Streptococcus pyogenes* and *Neisseria meningitidis*, the targeting domain of a first-order gRNA molecule is selected based on (1) distance from the target site (e.g., the start codon), such as within 500 bp (e.g., downstream) of the target site (e.g., the start codon), (2) a high level of orthogonality, and (3) the presence of 5'G. The targeting domain of a second-order gRNA molecule is selected based on (1) distance from the target site (e.g., the start codon), such as within 500 bp (e.g., downstream) of the target site (e.g., the start codon) and (2) a high level of orthogonality. The targeting domain of a third-order gRNA molecule is selected based on (1) distance from the target site (e.g., the start codon), such as within 500 bp (e.g., downstream) of the target site (e.g., the start codon) and (2) the presence of 5'G. The targeting domain of a level 4 gRNA molecule is selected based on (1) the distance from the target site (e.g., the start codon), for example, within 500 bp (e.g., downstream) of the target site (e.g., the start codon). The targeting domain of a level 5 gRNA molecule is selected based on the distance from the target site (e.g., the start codon), for example, within the range of the remaining coding sequence, for example, downstream of the first 500 bp of the coding sequence (e.g., anywhere from +500 (relative to the start codon) to the stop codon).

[0498] For Staphylococcus aureus, the targeting domain of a first-order gRNA molecule is selected based on (1) the distance from the target site (e.g., the start codon), for example, within 500 bp (e.g., downstream) of the target site (e.g., the start codon), (2) a high level of orthogonality, (3) the presence of 5'G, and (4) wherein the PAM is selected by NNGRRT (SEQ ID NO: 204). The targeting domain of a second-order gRNA molecule is selected based on (1) the distance from the target site (e.g., the start codon), for example, within 500 bp (e.g., downstream) of the target site (e.g., the start codon), (2) a high level of orthogonality, and (3) wherein the PAM is selected by NNGRRT. The targeting domain of a third-order gRNA molecule is selected based on (1) the distance from the target site (e.g., the start codon), for example, within 500 bp (e.g., downstream) of the target site (e.g., the start codon), and (2) wherein the PAM is selected by NNGRRT. The target domain of a level 4 gRNA molecule is selected based on (1) the distance from the target site (e.g., the start codon), for example, within 500 bp (e.g., downstream) of the target site (e.g., the start codon) and (2) where PAM is selected by NNGRRV. The target domain of a level 5 gRNA molecule is selected based on (1) the distance from the target site (e.g., the start codon), for example, within the remaining coding sequence, for example, downstream of the first 500 bp of the coding sequence (e.g., anywhere from +500 (relative to the start codon) to the stop codon), (2) the presence of 5'G, and (3) where PAM is selected by NNGRRT. The target domain of a level 6 gRNA molecule is selected based on (1) the distance from the target site (e.g., the start codon), for example, within the remaining coding sequence, for example, downstream of the first 500 bp of the coding sequence (e.g., anywhere from +500 (relative to the start codon) to the stop codon) and (2) where PAM is selected by NNGRRT. The target domains of the 7-level gRNA molecules are selected based on (1) distance from the target site (e.g., the start codon), for example, within the remaining coding sequence, such as downstream of the first 500 bp of the coding sequence (e.g., anywhere from +500 (relative to the start codon) to the stop codon) and (2) where PAM is NNGRRV. Note that the hierarchy is non-inclusive (listed only once per gRNA for this strategy). In some examples, no criteria for identifying gRNAs were used based on a specific hierarchy. Note that the hierarchy is non-inclusive (listed only once per target domain for this strategy). In some examples, no criteria for identifying target domains were used based on a specific hierarchy. The identified target domains are summarized in Table 1 below.

[0499] Table 1. Nucleotide sequences of the target domains of *Streptococcus pyogenes*, *Staphylococcus aureus*, and *Neisseria meningitidis* used to knock out the HSV-1 RS1 gene.

[0500]

[0501]

[0502] 6.2 Targeting domains for knocking out the HSV-2 RS1 gene

[0503] The target domains of gRNA used to knock out the HSV-2 RS1 gene in conjunction with the method disclosed in this invention were identified and graded as grade 5 for Streptococcus pyogenes, grade 7 for Staphylococcus aureus, and grade 5 for Neisseria meningitidis.

[0504] For *Streptococcus pyogenes* and *Neisseria meningitidis*, the targeting domain of a first-order gRNA molecule is selected based on (1) distance from the target site (e.g., the start codon), such as within 500 bp (e.g., downstream) of the target site (e.g., the start codon), (2) a high level of orthogonality, and (3) the presence of 5'G. The targeting domain of a second-order gRNA molecule is selected based on (1) distance from the target site (e.g., the start codon), such as within 500 bp (e.g., downstream) of the target site (e.g., the start codon) and (2) a high level of orthogonality. The targeting domain of a third-order gRNA molecule is selected based on (1) distance from the target site (e.g., the start codon), such as within 500 bp (e.g., downstream) of the target site (e.g., the start codon) and (2) the presence of 5'G. The targeting domain of a level 4 gRNA molecule is selected based on (1) the distance from the target site (e.g., the start codon), for example, within 500 bp (e.g., downstream) of the target site (e.g., the start codon). The targeting domain of a level 5 gRNA molecule is selected based on the distance from the target site (e.g., the start codon), for example, within the range of the remaining coding sequence, for example, downstream of the first 500 bp of the coding sequence (e.g., anywhere from +500 (relative to the start codon) to the stop codon).

[0505] For Staphylococcus aureus, the targeting domain of a first-order gRNA molecule is based on (1) the distance from the target site (e.g., the start codon), for example, within 500 bp (e.g., downstream) of the target site (e.g., the start codon), (2) a high level of orthogonality, (3) the presence of 5'G, and (4) where the PAM is selected by NNGRRT. The targeting domain of a second-order gRNA molecule is based on (1) the distance from the target site (e.g., the start codon), for example, within 500 bp (e.g., downstream) of the target site (e.g., the start codon), (2) a high level of orthogonality, and (3) where the PAM is selected by NNGRRT. The targeting domain of a third-order gRNA molecule is based on (1) the distance from the target site (e.g., the start codon), for example, within 500 bp (e.g., downstream) of the target site (e.g., the start codon), and (2) where the PAM is selected by NNGRRT. The target domain of a level 4 gRNA molecule is selected based on (1) the distance from the target site (e.g., the start codon), for example, within 500 bp (e.g., downstream) of the target site (e.g., the start codon) and (2) the PAM is selected by NNGRRV. The target domain of a level 5 gRNA molecule is selected based on (1) the distance from the target site (e.g., the start codon), for example, within the remaining coding sequence, for example, downstream of the first 500 bp of the coding sequence (e.g., anywhere from +500 (relative to the start codon) to the stop codon), (2) the presence of 5'G, and (3) where the PAM is selected by NNGRRT. The target domain of a level 6 gRNA molecule is selected based on (1) the distance from the target site (e.g., the start codon), for example, within the remaining coding sequence, for example, downstream of the first 500 bp of the coding sequence (e.g., from +500 (relative to the start codon) to the stop codon) and (2) where the PAM is selected by NNGRRT. The target domain of a level 7 gRNA molecule is selected based on (1) distance from the target site (e.g., the start codon), for example, within the remaining coding sequence, such as downstream of the first 500 bp of the coding sequence (e.g., +500 (relative to the start codon) to the stop codon) and (2) where PAM is NNGRRV. Note that the hierarchy is non-inclusive (listed only once per gRNA for this strategy). In some examples, the gRNA is not determined according to the criteria of a specific hierarchy.

[0506] Please note that the hierarchy is non-inclusive (each targeted domain is listed only once for this strategy). In some examples, the targeted domains were not determined according to the criteria for a specific hierarchy. The identified targeted domains are summarized in Table 2 below.

[0507] Table 2. Nucleotide sequences of the target domains of *Streptococcus pyogenes*, *Staphylococcus aureus*, and *Neisseria meningitidis* used to knock out the HSV-2 RS1 gene.

[0508]

[0509] 6.3 Targeting domains for knocking out the HSV-1 RL2 gene

[0510] The target domains of gRNA used to knock out the HSV-1 RL2 gene in conjunction with the method disclosed in this invention were identified and graded as grade 5 for Streptococcus pyogenes, grade 7 for Staphylococcus aureus, and grade 5 for Neisseria meningitidis.

[0511] For *Streptococcus pyogenes* and *Neisseria meningitidis*, the targeting domain of a first-order gRNA molecule is selected based on (1) distance from the target site (e.g., the start codon), such as within 500 bp (e.g., downstream) of the target site (e.g., the start codon), (2) a high level of orthogonality, and (3) the presence of 5'G. The targeting domain of a second-order gRNA molecule is selected based on (1) distance from the target site (e.g., the start codon), such as within 500 bp (e.g., downstream) of the target site (e.g., the start codon) and (2) a high level of orthogonality. The targeting domain of a third-order gRNA molecule is selected based on (1) distance from the target site (e.g., the start codon), such as within 500 bp (e.g., downstream) of the target site (e.g., the start codon) and (2) the presence of 5'G. The targeting domain of a level 4 gRNA molecule is selected based on (1) the distance from the target site (e.g., the start codon), for example, within 500 bp (e.g., downstream) of the target site (e.g., the start codon). The targeting domain of a level 5 gRNA molecule is selected based on the distance from the target site (e.g., the start codon), for example, within the range of the remaining coding sequence, for example, downstream of the first 500 bp of the coding sequence (e.g., anywhere from +500 (relative to the start codon) to the stop codon).

[0512] For Staphylococcus aureus, the targeting domain of a first-order gRNA molecule is based on (1) the distance from the target site (e.g., the start codon), for example, within 500 bp (e.g., downstream) of the target site (e.g., the start codon), (2) a high level of orthogonality, (3) the presence of 5'G, and (4) where the PAM is selected by NNGRRT. The targeting domain of a second-order gRNA molecule is based on (1) the distance from the target site (e.g., the start codon), for example, within 500 bp (e.g., downstream) of the target site (e.g., the start codon), (2) a high level of orthogonality, and (3) where the PAM is selected by NNGRRT. The targeting domain of a third-order gRNA molecule is based on (1) the distance from the target site (e.g., the start codon), for example, within 500 bp (e.g., downstream) of the target site (e.g., the start codon), and (2) where the PAM is selected by NNGRRT. The target domain of a level 4 gRNA molecule is selected based on (1) the distance from the target site (e.g., the start codon), for example, within 500 bp (e.g., downstream) of the target site (e.g., the start codon) and (2) where PAM is selected by NNGRRV. The target domain of a level 5 gRNA molecule is selected based on (1) the distance from the target site (e.g., the start codon), for example, within the remaining coding sequence, for example, downstream of the first 500 bp of the coding sequence (e.g., anywhere from +500 (relative to the start codon) to the stop codon), (2) the presence of 5'G, and (3) where PAM is selected by NNGRRT. The target domain of a level 6 gRNA molecule is selected based on (1) the distance from the target site (e.g., the start codon), for example, within the remaining coding sequence, for example, downstream of the first 500 bp of the coding sequence (e.g., anywhere from +500 (relative to the start codon) to the stop codon) and (2) where PAM is selected by NNGRRT. The targeting domains of the 7-level gRNA molecules are selected based on (1) distance from the target site (e.g., the start codon), for example, within the remaining coding sequence, such as downstream of the first 500 bp of the coding sequence (e.g., anywhere from +500 (relative to the start codon) to the stop codon) and (2) where PAM is NNGRRV. Note that the levels are non-inclusive (listed only once per gRNA for this strategy). In some examples, the gRNA is not determined according to the criteria for a specific level.

[0513] Please note that the hierarchy is non-inclusive (the policy is listed only once for each target domain). In some examples, the target domains were not determined based on criteria for a specific hierarchy. The identified target domains are summarized in Table 3 below.

[0514] Table 3. Nucleotide sequences of the target domains of *Streptococcus pyogenes*, *Staphylococcus aureus*, and *Neisseria meningitidis* used to knock out the HSV-1 RL2 gene.

[0515]

[0516] 6.4 Targeting domains for knocking out the HSV-2 RL2 gene

[0517] The target domains of gRNA used to knock out the HSV-2 RL2 gene in conjunction with the method disclosed in this invention were identified and graded as grade 5 for Streptococcus pyogenes, grade 7 for Staphylococcus aureus, and grade 5 for Neisseria meningitidis.

[0518] For *Streptococcus pyogenes* and *Neisseria meningitidis*, the targeting domain of a primary gRNA molecule is selected based on (1) distance from the target site (e.g., the start codon), such as within 500 bp (e.g., downstream) of the target site (e.g., the start codon), (2) a high level of orthogonality, and (3) the presence of 5'G. The targeting domain of a secondary gRNA molecule is selected based on (1) distance from the target site (e.g., the start codon), such as within 500 bp (e.g., downstream) of the target site (e.g., the start codon) and (2) a high level of orthogonality. The targeting domain of a primary gRNA molecule is selected based on (1) distance from the target site (e.g., the start codon), such as within 500 bp (e.g., downstream) of the target site (e.g., the start codon) and (2) the presence of 5'G. The targeting domain of a level 4 gRNA molecule is selected based on (1) the distance from the target site (e.g., the start codon), for example, within 500 bp (e.g., downstream) of the target site (e.g., the start codon). The targeting domain of a level 5 gRNA molecule is selected based on the distance from the target site (e.g., the start codon), for example, within the range of the remaining coding sequence, for example, downstream of the first 500 bp of the coding sequence (e.g., anywhere from +500 (relative to the start codon) to the stop codon).

[0519] For Staphylococcus aureus, the targeting domain of a first-order gRNA molecule is based on (1) the distance from the target site (e.g., the start codon), for example, within 500 bp (e.g., downstream) of the target site (e.g., the start codon), (2) a high level of orthogonality, (3) the presence of 5'G, and (4) where the PAM is selected by NNGRRT. The targeting domain of a second-order gRNA molecule is based on (1) the distance from the target site (e.g., the start codon), for example, within 500 bp (e.g., downstream) of the target site (e.g., the start codon), (2) a high level of orthogonality, and (3) where the PAM is selected by NNGRRT. The targeting domain of a third-order gRNA molecule is based on (1) the distance from the target site (e.g., the start codon), for example, within 500 bp (e.g., downstream) of the target site (e.g., the start codon), and (2) where the PAM is selected by NNGRRT. The target domain of a level 4 gRNA molecule is selected based on (1) the distance from the target site (e.g., the start codon), for example, within 500 bp (e.g., downstream) of the target site (e.g., the start codon) and (2) where PAM is selected by NNGRRV. The target domain of a level 5 gRNA molecule is selected based on (1) the distance from the target site (e.g., the start codon), for example, within the remaining coding sequence, for example, downstream of the first 500 bp of the coding sequence (e.g., anywhere from +500 (relative to the start codon) to the stop codon), (2) the presence of 5'G, and (3) where PAM is selected by NNGRRT. The target domain of a level 6 gRNA molecule is selected based on (1) the distance from the target site (e.g., the start codon), for example, within the remaining coding sequence, for example, downstream of the first 500 bp of the coding sequence (e.g., anywhere from +500 (relative to the start codon) to the stop codon) and (2) where PAM is selected by NNGRRT. The target domain of a level 7 gRNA molecule is selected based on (1) distance from the target site (e.g., the start codon), for example, within the remaining coding sequence, such as downstream of the first 500 bp of the coding sequence (e.g., anywhere from +500 (relative to the start codon) to the stop codon) and (2) where PAM is NNGRRV. Note that the hierarchy is non-inclusive (the strategy is listed only once per gRNA). In some examples, the gRNA is not determined according to the criteria of a specific hierarchy.

[0520] Please note that the hierarchy is non-inclusive (each targeted domain is listed only once for this strategy). In some examples, the targeted domains were not determined according to the criteria for a specific hierarchy. The identified targeted domains are summarized in Table 4 below.

[0521] Table 4. Nucleotide sequences of the target domains of *Streptococcus pyogenes*, *Staphylococcus aureus*, and *Neisseria meningitidis* used to knock out the HSV-2 RL2 gene.

[0522]

[0523] 6.5 Targeting domains for knocking out HSV-1 LAT introns

[0524] The target domains in gRNA used to knock out the HSV-1 LAT introns in conjunction with the method disclosed in this invention were identified and graded as grade 5 for Streptococcus pyogenes, grade 7 for Staphylococcus aureus, and grade 5 for Neisseria meningitidis.

[0525] For *Streptococcus pyogenes* and *Neisseria meningitidis*, the targeting domains of first-order gRNA molecules are selected based on (1) distance to the target site, e.g., within the first 500 bp of the LAT intron, (2) a high level of orthogonality, and (3) the presence of 5'G. The targeting domains of second-order gRNA molecules are selected based on (1) distance to the target site, e.g., within the first 500 bp of the LAT intron and (2) a high level of orthogonality. The targeting domains of third-order gRNA molecules are selected based on (1) distance to the target site, e.g., within the first 500 bp of the LAT intron and (2) the presence of 5'G. The targeting domains of fourth-order gRNA molecules are selected based on distance to the target site, e.g., within the first 500 bp of the LAT intron. The targeting domains of fifth-order gRNA molecules are selected based on distance to the target site, e.g., within the remaining sequence of the LAT intron, e.g., downstream of the first 500 bp of the LAT intron.

[0526] For Staphylococcus aureus, the targeting domain of first-order gRNA molecules is based on (1) the distance to the target site, e.g., within the first 500 bp of the LAT intron, (2) a high level of orthogonality, (3) the presence of 5'G, and (4) where PAM is selected by NNGRRT. The targeting domain of second-order gRNA molecules is based on (1) the distance to the target site, e.g., within the first 500 bp of the LAT intron, (2) a high level of orthogonality, and (3) where PAM is selected by NNGRRT. The targeting domain of third-order gRNA molecules is based on (1) the distance to the target site, e.g., within the first 500 bp of the LAT intron and (2) where PAM is selected by NNGRRT. The targeting domain of fourth-order gRNA molecules is based on (1) the distance to the target site, e.g., within the first 500 bp of the LAT intron and (2) where PAM is selected by NNGRRV. The targeting domain of level 5 gRNA molecules is based on (1) the distance to the target site, such as within the remaining sequence of the LAT intron, such as downstream of the first 500 bp of the LAT intron, (2) the presence of 5'G, and (3) where PAM is selected by NNGRRT. The targeting domain of level 6 gRNA molecules is based on (1) the distance to the target site, such as within the remaining sequence of the LAT intron, such as downstream of the first 500 bp of the LAT intron, and (2) where PAM is selected by NNGRRT. The targeting domain of level 7 gRNA molecules is based on (1) the distance to the target site, such as within the remaining sequence of the LAT intron, such as downstream of the first 500 bp of the LAT intron, and (2) where PAM is selected by NNGRRV. Note that the hierarchy is not included (the strategy is listed only once per gRNA). In some examples, the gRNA was not determined according to the criteria for a specific hierarchy.

[0527] Please note that the hierarchy is non-inclusive (each targeted domain is listed only once for this strategy). In some examples, the targeted domains were not determined based on the criteria for a specific hierarchy. The identified targeted domains are summarized in Table 5 below.

[0528] Table 5. Nucleotide sequences of the target domains of Streptococcus pyogenes, Staphylococcus aureus, and Neisseria meningitidis used to knock out the HSV-1 LAT intron.

[0529]

[0530]

[0531] 6.6 Targeting domains for knocking out HSV-2LAT introns

[0532] The target domains of the HSV-2LAT intron gene, which are combined with the method disclosed in this invention to knock out the HSV-2LAT intron gene, were identified and graded as grade 5 for Streptococcus pyogenes, grade 7 for Staphylococcus aureus, and grade 5 for Neisseria meningitidis.

[0533] For *Streptococcus pyogenes* and *Neisseria meningitidis*, the targeting domains of first-order gRNA molecules are selected based on (1) distance to the target site, e.g., within the first 500 bp of the LAT intron, (2) a high level of orthogonality, and (3) the presence of 5'G. The targeting domains of second-order gRNA molecules are selected based on (1) distance to the target site, e.g., within the first 500 bp of the LAT intron and (2) a high level of orthogonality. The targeting domains of third-order gRNA molecules are selected based on (1) distance to the target site, e.g., within the first 500 bp of the LAT intron and (2) the presence of 5'G. The targeting domains of fourth-order gRNA molecules are selected based on distance to the target site, e.g., within the first 500 bp of the LAT intron. The targeting domains of fifth-order gRNA molecules are selected based on distance to the target site, e.g., within the remaining sequence of the LAT intron, e.g., downstream of the first 500 bp of the LAT intron.

[0534] For Staphylococcus aureus, the targeting domain of first-order gRNA molecules is based on (1) the distance to the target site, e.g., within the first 500 bp of the LAT intron, (2) a high level of orthogonality, (3) the presence of 5'G, and (4) where PAM is selected by NNGRRT. The targeting domain of second-order gRNA molecules is based on (1) the distance to the target site, e.g., within the first 500 bp of the LAT intron, (2) a high level of orthogonality, and (3) where PAM is selected by NNGRRT. The targeting domain of third-order gRNA molecules is based on (1) the distance to the target site, e.g., within the first 500 bp of the LAT intron and (2) where PAM is selected by NNGRRT. The targeting domain of fourth-order gRNA molecules is based on (1) the distance to the target site, e.g., within the first 500 bp of the LAT intron and (2) where PAM is selected by NNGRRV. The targeting domains of level 5 gRNA molecules are based on (1) the distance to the target site, such as within the remaining sequence of the LAT intron, such as downstream of the first 500 bp of the LAT intron, (2) the presence of 5'G, and (3) where PAM is selected by NNGRRT. The targeting domains of level 6 gRNA molecules are based on (1) the distance to the target site, such as within the remaining sequence of the LAT intron, such as downstream of the first 500 bp of the LAT intron, and (2) where PAM is selected by NNGRRT. The targeting domains of level 7 gRNA molecules are based on (1) the distance to the target site, such as within the remaining sequence of the LAT intron, such as downstream of the first 500 bp of the LAT intron, and (2) where PAM is selected by NNGRRV. Note that the hierarchy is non-inclusive (each gRNA is listed only once for this strategy). In some examples, the gRNA was not determined according to the criteria for a specific hierarchy.

[0535] Please note that the hierarchy is non-inclusive (each targeted domain is listed only once for this strategy). In some examples, the targeted domains were not determined according to the criteria for a specific hierarchy. The identified targeted domains are summarized in Table 6 below.

[0536] Table 6 shows the nucleotide sequences of the target domains of Streptococcus pyogenes, Staphylococcus aureus, and Neisseria meningitidis used to knock out the HSV-2LAT intron.

[0537]

[0538] 6.7 Targeting domains for knocking down the HSV-1 RS1 gene

[0539] The target domains of the HSV-1 RS1 gene, which are combined with the method disclosed in this invention and used to knock down the HSV-1 RS1 gene in gRNA, were identified and graded as grade 5 for Streptococcus pyogenes, grade 7 for Staphylococcus aureus, and grade 5 for Neisseria meningitidis.

[0540] For *Streptococcus pyogenes* and *Neisseria meningitidis*, the targeting domains of first-order gRNA molecules are selected based on (1) distance from the target site, such as within 500 bp (e.g., upstream or downstream) of the transcription start site (TSS), (2) a high level of orthogonality, and (3) the presence of 5'G. The targeting domains of second-order gRNAs are selected based on (1) distance from the target site, such as within 500 bp (e.g., upstream or downstream) of the transcription start site (TSS) and (2) a high level of orthogonality. The targeting domains of third-order gRNA molecules are selected based on (1) distance from the target site, such as within 500 bp (e.g., upstream or downstream) of the transcription start site (TSS) and (2) the presence of 5'G. The targeting domains of fourth-order gRNAs are selected based on distance from the target site, such as within 500 bp (e.g., upstream or downstream) of the transcription start site (TSS). The targeting domain of a level 5 gRNA molecule is selected based on the distance to the target site, such as within an additional 500 bp (e.g., upstream or downstream) of the transcription start site (TSS), for example, extending 1 kb upstream and downstream of the TSS.

[0541] For Staphylococcus aureus, the targeting domain of a first-order gRNA molecule is selected based on (1) distance from the target site, e.g., within 500 bp of the transcription start site (TSS) (e.g., upstream or downstream), (2) a high level of orthogonality, (3) the presence of 5'G, and (4) PAM is selected by NNGRRT. The targeting domain of a second-order gRNA molecule is selected based on (1) distance from the target site, e.g., within 500 bp of the transcription start site (TSS) (e.g., upstream or downstream), (2) a high level of orthogonality, and (3) PAM is selected by NNGRRT. The targeting domain of a third-order gRNA molecule is selected based on (1) distance from the target site, e.g., within 500 bp of the transcription start site (TSS) (e.g., upstream or downstream) and (2) PAM is selected by NNGRRT. The targeting domain of a level 4 gRNA molecule is based on (1) the distance to the target site, for example, within 500 bp of the transcription start site (TSS) (e.g., upstream or downstream) and (2) where the PAM is selected by NNGRRV. The targeting domain of a level 5 gRNA molecule is based on (1) the distance to the target site, for example, within an additional 500 bp of the transcription start site (TSS) (e.g., upstream or downstream), for example, extending 1 kb upstream and downstream of the TSS, (2) the presence of 5'G, and (3) where the PAM is selected by NNGRRT. The targeting domain of a level 6 gRNA molecule is based on (1) the distance to the target site, for example, within an additional 500 bp of the transcription start site (TSS) (e.g., upstream or downstream), for example, extending 1 kb upstream and downstream of the TSS, and (2) the PAM is selected by NNGRRT. The targeting domains of the 7-level gRNA molecules are selected based on (1) the distance to the target site, such as within an additional 500 bp (e.g., upstream or downstream) of the transcription start site (TSS), for example, extending 1 kb upstream and downstream of the TSS, and (2) the PAM is selected by NNGRRV. Note that the hierarchy is non-inclusive (each gRNA is listed only once for this strategy). In some examples, the gRNA is not determined according to the criteria of a specific hierarchy.

[0542] Please note that the hierarchy is non-inclusive (each targeted domain is listed only once for this strategy). In some examples, the targeted domains were not determined based on criteria for a specific hierarchy. The identified targeted domains are summarized in Table 7 below.

[0543] Table 7. Nucleotide sequences of the target domains of *Streptococcus pyogenes*, *Staphylococcus aureus*, and *Neisseria meningitidis* used to knock down the HSV-1 RS1 gene.

[0544]

[0545] 6.8 Targeting domains for knocking down the HSV-2 RS1 gene

[0546] The target domains of the HSV-2 RS1 gene, which are combined with the method disclosed in this invention and used to knock down the HSV-2 RS1 gene in gRNA, were identified and graded as grade 5 for Streptococcus pyogenes, grade 7 for Staphylococcus aureus, and grade 5 for Neisseria meningitidis.

[0547] For *Streptococcus pyogenes* and *Neisseria meningitidis*, the targeting domain of a first-order gRNA is selected based on (1) distance from the target site, such as within 500 bp (e.g., upstream or downstream) of the start codon, (2) a high level of orthogonality, and (3) the presence of 5'G. The targeting domain of a second-order gRNA is selected based on (1) distance from the target site, such as within 500 bp (e.g., upstream or downstream) of the start codon and (2) a high level of orthogonality. The targeting domain of a third-order gRNA is selected based on (1) distance from the target site, such as within 500 bp (e.g., upstream or downstream) of the start codon and (2) the presence of 5'G. The targeting domain of a fourth-order gRNA is selected based on distance from the target site, such as within 500 bp (e.g., upstream or downstream) of the start codon. The targeting domain of a level 5 gRNA molecule is selected based on the distance to the target site, such as within 500 bp (e.g., upstream or downstream) of the start codon, for example, extending 1 kb upstream and downstream of the start codon.

[0548] For Staphylococcus aureus, the targeting domain of a first-order gRNA molecule is selected based on (1) distance from the target site, such as within 500 bp of the start codon (e.g., upstream or downstream), (2) a high level of orthogonality, (3) the presence of 5'G, and (4) PAM is selected by NNGRRT. The targeting domain of a second-order gRNA molecule is selected based on (1) distance from the target site, such as within 500 bp of the start codon (e.g., upstream or downstream), (2) a high level of orthogonality, and (3) PAM is selected by NNGRRT. The targeting domain of a third-order gRNA molecule is selected based on (1) distance from the target site, such as within 500 bp of the start codon (e.g., upstream or downstream) and (2) PAM is selected by NNGRRT. The targeting domain of a fourth-order gRNA molecule is selected based on (1) distance from the target site, such as within 500 bp of the start codon (e.g., upstream or downstream) and (2) PAM is selected by NNGRRV. The targeting domain of level 5 gRNA molecules is based on (1) the distance to the target site, such as within an additional 500 bp (e.g., upstream or downstream) of the start codon, such as extending 1 kb upstream and downstream of the start codon, (2) the presence of 5'G, and (3) where PAM is selected by NNGRRT. The targeting domain of level 6 gRNA molecules is based on (1) the distance to the target site, such as within an additional 500 bp (e.g., upstream or downstream) of the start codon, such as extending 1 kb upstream and downstream of the start codon, and (2) where PAM is selected by NNGRRT. The targeting domain of level 7 gRNA molecules is based on (1) the distance to the target site, such as within an additional 500 bp (e.g., upstream or downstream) of the start codon, such as extending 1 kb upstream and downstream of the start codon, and (2) where PAM is selected by NNGRRV. Note that the hierarchy is non-inclusive (each gRNA is listed only once for this strategy). In some examples, the gRNA was not determined according to the criteria for a specific hierarchy.

[0549] Please note that the hierarchy is non-inclusive (each targeted domain is listed only once for this strategy). In some examples, the targeted domains were not determined according to the criteria for a specific hierarchy. The identified targeted domains are summarized in Table 8 below.

[0550] Table 8. Nucleotide sequences of the target domains of *Streptococcus pyogenes*, *Staphylococcus aureus*, and *Neisseria meningitidis* used to knock down the HSV-2 RS1 gene.

[0551]

[0552]

[0553] 6.9 Targeting domain for knocking down the HSV-1 RL2 gene

[0554] The target domains of gRNA used to knock down the HSV-1 RL2 gene in conjunction with the method disclosed in this invention were identified and graded as grade 5 for Streptococcus pyogenes, grade 7 for Staphylococcus aureus, and grade 5 for Neisseria meningitidis.

[0555] For *Streptococcus pyogenes* and *Neisseria meningitidis*, the targeting domains of first-order gRNA molecules are selected based on (1) distance from the target site, such as within 500 bp (e.g., upstream or downstream) of the transcription start site (TSS), (2) a high level of orthogonality, and (3) the presence of 5'G. The targeting domains of second-order gRNAs are selected based on (1) distance from the target site, such as within 500 bp (e.g., upstream or downstream) of the transcription start site (TSS) and (2) a high level of orthogonality. The targeting domains of third-order gRNA molecules are selected based on (1) distance from the target site, such as within 500 bp (e.g., upstream or downstream) of the transcription start site (TSS) and (2) the presence of 5'G. The targeting domains of fourth-order gRNAs are selected based on distance from the target site, such as within 500 bp (e.g., upstream or downstream) of the transcription start site (TSS). The targeting domain of a level 5 gRNA molecule is selected based on the distance to the target site, such as within an additional 500 bp (e.g., upstream or downstream) of the transcription start site (TSS), for example, extending 1 kb upstream and downstream of the TSS.

[0556] For Staphylococcus aureus, the targeting domain of a first-order gRNA molecule is selected based on (1) distance from the target site, e.g., within 500 bp of the transcription start site (TSS) (e.g., upstream or downstream), (2) a high level of orthogonality, (3) the presence of 5'G, and (4) PAM is selected by NNGRRT. The targeting domain of a second-order gRNA molecule is selected based on (1) distance from the target site, e.g., within 500 bp of the transcription start site (TSS) (e.g., upstream or downstream), (2) a high level of orthogonality, and (3) PAM is selected by NNGRRT. The targeting domain of a third-order gRNA molecule is selected based on (1) distance from the target site, e.g., within 500 bp of the transcription start site (TSS) (e.g., upstream or downstream) and (2) PAM is selected by NNGRRT. The targeting domain of a level 4 gRNA molecule is based on (1) the distance to the target site, for example, within 500 bp of the transcription start site (TSS) (e.g., upstream or downstream) and (2) where the PAM is selected by NNGRRV. The targeting domain of a level 5 gRNA molecule is based on (1) the distance to the target site, for example, within an additional 500 bp of the transcription start site (TSS) (e.g., upstream or downstream), for example, extending 1 kb upstream and downstream of the TSS, (2) the presence of 5'G, and (3) where the PAM is selected by NNGRRT. The targeting domain of a level 6 gRNA molecule is based on (1) the distance to the target site, for example, within an additional 500 bp of the transcription start site (TSS) (e.g., upstream or downstream), for example, extending 1 kb upstream and downstream of the TSS, and (2) where the PAM is selected by NNGRRT. The targeting domains of 7-level gRNA molecules are based on (1) the distance to the target site, such as within an additional 500 bp (e.g., upstream or downstream) of the transcription start site (TSS), for example, extending 1 kb upstream and downstream of the TSS, and (2) where PAM is selected by NNGRRV. Note that the hierarchy is non-inclusive (each gRNA is listed only once for this strategy). In some examples, the gRNA is not determined according to the criteria of a specific hierarchy.

[0557] Please note that the hierarchy is non-inclusive (each targeted domain is listed only once for this strategy). In some examples, the targeted domains were not determined based on criteria for a specific hierarchy. The identified targeted domains are summarized in Table 9 below.

[0558] Table 9. Nucleotide sequences of the target domains of *Streptococcus pyogenes*, *Staphylococcus aureus*, and *Neisseria meningitidis* used to knock down the HSV-1 RL2 gene.

[0559]

[0560] 6.10 Targeting domain for knocking down the HSV-2 RL2 gene

[0561] The target domains in gRNAs used to knock down the HSV-2 RL2 gene in conjunction with the methods disclosed in this invention were identified and graded into five levels for *Streptococcus pyogenes*, seven levels for *Staphylococcus aureus*, and five levels for *Neisseria meningitidis*. For *Streptococcus pyogenes* and *Neisseria meningitidis*, the target domains of the grade 1 gRNA molecules were selected based on (1) distance from the target site, e.g., within 500 bp (e.g., upstream or downstream) of the start site (TSS), (2) a high level of orthogonality, and (3) the presence of 5'G. The target domains of the grade 2 gRNA molecules were selected based on (1) distance from the target site, e.g., within 500 bp (e.g., upstream or downstream) of the start site (TSS) and (2) a high level of orthogonality. The target domains of the grade 3 gRNA molecules were selected based on (1) distance from the target site, e.g., within 500 bp (e.g., upstream or downstream) of the start codon and (2) the presence of 5'G. The targeting domain of level 4 gRNAs is selected based on the distance to the target site, such as within 500 bp (e.g., upstream or downstream) of the start codon. The targeting domain of level 5 gRNA molecules is selected based on the distance to the target site, such as within an additional 500 bp (e.g., upstream or downstream) of the start codon, for example, extending 1 kb upstream and downstream of the start codon.

[0562] For Staphylococcus aureus, the targeting domain of a first-order gRNA molecule is selected based on (1) distance from the target site, such as within 500 bp of the start codon (e.g., upstream or downstream), (2) a high level of orthogonality, (3) the presence of 5'G, and (4) PAM is selected by NNGRRT. The targeting domain of a second-order gRNA molecule is selected based on (1) distance from the target site, such as within 500 bp of the start codon (e.g., upstream or downstream), (2) a high level of orthogonality, and (3) PAM is selected by NNGRRT. The targeting domain of a third-order gRNA molecule is selected based on (1) distance from the target site, such as within 500 bp of the start codon (e.g., upstream or downstream) and (2) PAM is selected by NNGRRT. The targeting domain of a fourth-order gRNA molecule is selected based on (1) distance from the target site, such as within 500 bp of the start codon (e.g., upstream or downstream) and (2) PAM is selected by NNGRRV. The targeting domain of level 5 gRNA molecules is based on (1) the distance to the target site, such as within an additional 500 bp (e.g., upstream or downstream) of the start codon, such as extending 1 kb upstream and downstream of the start codon, (2) the presence of 5'G, and (3) where PAM is selected by NNGRRT. The targeting domain of level 6 gRNA molecules is based on (1) the distance to the target site, such as within an additional 500 bp (e.g., upstream or downstream) of the start codon, such as extending 1 kb upstream and downstream of the start codon, and (2) where PAM is selected by NNGRRT. The targeting domain of level 7 gRNA molecules is based on (1) the distance to the target site, such as within an additional 500 bp (e.g., upstream or downstream) of the start codon, such as extending 1 kb upstream and downstream of the start codon, and (2) where PAM is selected by NNGRRV. Note that the hierarchy is non-inclusive (each gRNA is listed only once for this strategy). In some examples, the gRNA was not determined according to the criteria for a specific hierarchy.

[0563] Please note that the hierarchy is non-inclusive (each targeted domain is listed only once for this strategy). In some examples, the targeted domains were not determined based on the criteria for a specific hierarchy. The identified targeted domains are summarized in Table 10 below.

[0564] Table 10. Nucleotide sequences of the target domains of *Streptococcus pyogenes*, *Staphylococcus aureus*, and *Neisseria meningitidis* used to knock down the HSV-2 RL2 gene.

[0565]

[0566] 6.11 Targeting domain for knocking down the HSV-1 LAT gene

[0567] The target domains of the HSV-1LAT gene in gRNA, which are combined with the method disclosed in this invention, for knocking down the HSV-1LAT gene, were identified and graded as grade 5 for Streptococcus pyogenes, grade 7 for Staphylococcus aureus, and grade 5 for Neisseria meningitidis.

[0568] For *Streptococcus pyogenes* and *Neisseria meningitidis*, the targeting domains of first-order gRNA molecules are selected based on (1) distance from the target site, such as within 500 bp (e.g., upstream or downstream) of the transcription start site (TSS), (2) a high level of orthogonality, and (3) the presence of 5'G. The targeting domains of second-order gRNAs are selected based on (1) distance from the target site, such as within 500 bp (e.g., upstream or downstream) of the transcription start site (TSS) and (2) a high level of orthogonality. The targeting domains of third-order gRNA molecules are selected based on (1) distance from the target site, such as within 500 bp (e.g., upstream or downstream) of the transcription start site (TSS) and (2) the presence of 5'G. The targeting domains of fourth-order gRNAs are selected based on distance from the target site, such as within 500 bp (e.g., upstream or downstream) of the transcription start site (TSS). The targeting domain of a level 5 gRNA molecule is selected based on the distance to the target site, such as within an additional 500 bp (e.g., upstream or downstream) of the transcription start site (TSS), for example, extending 1 kb upstream and downstream of the TSS.

[0569] For Staphylococcus aureus, the targeting domain of a first-order gRNA molecule is selected based on (1) distance from the target site, e.g., within 500 bp of the transcription start site (TSS) (e.g., upstream or downstream), (2) a high level of orthogonality, (3) the presence of 5'G, and (4) PAM is selected by NNGRRT. The targeting domain of a second-order gRNA molecule is selected based on (1) distance from the target site, e.g., within 500 bp of the transcription start site (TSS) (e.g., upstream or downstream), (2) a high level of orthogonality, and (3) PAM is selected by NNGRRT. The targeting domain of a third-order gRNA molecule is selected based on (1) distance from the target site, e.g., within 500 bp of the transcription start site (TSS) (e.g., upstream or downstream) and (2) PAM is selected by NNGRRT. The targeting domain of a level 4 gRNA molecule is based on (1) the distance to the target site, for example, within 500 bp of the transcription start site (TSS) (e.g., upstream or downstream) and (2) where the PAM is selected by NNGRRV. The targeting domain of a level 5 gRNA molecule is based on (1) the distance to the target site, for example, within an additional 500 bp of the transcription start site (TSS) (e.g., upstream or downstream), for example, extending 1 kb upstream and downstream of the TSS, (2) the presence of 5'G, and (3) where the PAM is selected by NNGRRT. The targeting domain of a level 6 gRNA molecule is based on (1) the distance to the target site, for example, within an additional 500 bp of the transcription start site (TSS) (e.g., upstream or downstream), for example, extending 1 kb upstream and downstream of the TSS, and (2) where the PAM is selected by NNGRRT. The targeting domains of 7-level gRNA molecules are based on (1) the distance to the target site, such as within an additional 500 bp (e.g., upstream or downstream) of the transcription start site (TSS), for example, extending 1 kb upstream and downstream of the TSS, and (2) where PAM is selected by NNGRRV. Note that the hierarchy is non-inclusive (each gRNA is listed only once for this strategy). In some examples, the gRNA is not determined according to the criteria of a specific hierarchy.

[0570] Please note that the hierarchy is non-inclusive (each targeted domain is listed only once for this strategy). In some examples, the targeted domains were not determined based on the criteria for a specific hierarchy. The identified targeted domains are summarized in Table 11 below.

[0571] Table 11 Nucleotide sequences of the target domains of *Streptococcus pyogenes*, *Staphylococcus aureus*, and *Neisseria meningitidis* used to knock down the HSV-1 LAT gene.

[0572]

[0573]

[0574] 6.12 Targeting domain for knocking down the HSV-2 LAT gene

[0575] The target domains of the HSV-2LAT gene that are combined with the method disclosed in this invention in gRNA for knocking down the gene are identified and graded as grade 5 for Streptococcus pyogenes, grade 7 for Staphylococcus aureus, and grade 5 for Neisseria meningitidis.

[0576] For *Streptococcus pyogenes* and *Neisseria meningitidis*, the targeting domains of first-order gRNA molecules are selected based on (1) distance from the target site, such as within 500 bp (e.g., upstream or downstream) of the transcription start site (TSS), (2) a high level of orthogonality, and (3) the presence of 5'G. The targeting domains of second-order gRNAs are selected based on (1) distance from the target site, such as within 500 bp (e.g., upstream or downstream) of the transcription start site (TSS) and (2) a high level of orthogonality. The targeting domains of third-order gRNA molecules are selected based on (1) distance from the target site, such as within 500 bp (e.g., upstream or downstream) of the transcription start site (TSS) and (2) the presence of 5'G. The targeting domains of fourth-order gRNAs are selected based on distance from the target site, such as within 500 bp (e.g., upstream or downstream) of the transcription start site (TSS). The targeting domain of a level 5 gRNA molecule is selected based on the distance to the target site, such as within an additional 500 bp (e.g., upstream or downstream) of the transcription start site (TSS), for example, extending 1 kb upstream and downstream of the TSS.

[0577] For Staphylococcus aureus, the targeting domain of a first-order gRNA molecule is selected based on (1) distance from the target site, e.g., within 500 bp of the transcription start site (TSS) (e.g., upstream or downstream), (2) a high level of orthogonality, (3) the presence of 5'G, and (4) PAM is selected by NNGRRT. The targeting domain of a second-order gRNA molecule is selected based on (1) distance from the target site, e.g., within 500 bp of the transcription start site (TSS) (e.g., upstream or downstream), (2) a high level of orthogonality, and (3) PAM is selected by NNGRRT. The targeting domain of a third-order gRNA molecule is selected based on (1) distance from the target site, e.g., within 500 bp of the transcription start site (TSS) (e.g., upstream or downstream) and (2) PAM is selected by NNGRRT. The targeting domain of a level 4 gRNA molecule is based on (1) the distance to the target site, for example, within 500 bp of the transcription start site (TSS) (e.g., upstream or downstream) and (2) where the PAM is selected by NNGRRV. The targeting domain of a level 5 gRNA molecule is based on (1) the distance to the target site, for example, within an additional 500 bp of the transcription start site (TSS) (e.g., upstream or downstream), for example, extending 1 kb upstream and downstream of the TSS, (2) the presence of 5'G, and (3) where the PAM is selected by NNGRRT. The targeting domain of a level 6 gRNA molecule is based on (1) the distance to the target site, for example, within an additional 500 bp of the transcription start site (TSS) (e.g., upstream or downstream), for example, extending 1 kb upstream and downstream of the TSS, and (2) where the PAM is selected by NNGRRT. The targeting domains of 7-level gRNA molecules are based on (1) the distance to the target site, such as within an additional 500 bp (e.g., upstream or downstream) of the transcription start site (TSS), for example, extending 1 kb upstream and downstream of the TSS, and (2) where PAM is selected by NNGRRV. Note that the hierarchy is non-inclusive (each gRNA is listed only once for this strategy). In some examples, the gRNA is not determined according to the criteria of a specific hierarchy.

[0578] Please note that the hierarchy is non-inclusive (each targeted domain is listed only once for this strategy). In some examples, the targeted domains were not determined based on the criteria for a specific hierarchy. The identified targeted domains are summarized in Table 12 below.

[0579] Table 12 Nucleotide sequences of the target domains of *Streptococcus pyogenes*, *Staphylococcus aureus*, and *Neisseria meningitidis* used to knock down the HSV-2LAT gene.

[0580]

[0581] One or more gRNA molecules described in this invention (e.g., gRNA molecules including the target domains described in Tables 1-12) can be used with at least one Cas9 molecule (e.g., Streptococcus pyogenes Cas9 molecule and / or Staphylococcus aureus Cas9 molecule) to form single-stranded or double-stranded cuts, for example, by using a Cas9 nickase molecule to generate single-stranded breaks or by using a Cas9 nuclease molecule to generate double-stranded breaks.

[0582] In some implementations, when a single gRNA molecule is used to target the Cas9 nickase to generate a single-strand break near the RS1, RL2, or LAT target, for example, the gRNA is used to target upstream (e.g., within 500 bp upstream) or downstream (e.g., within 500 bp downstream) of the RS1, RL2, or LAT target.

[0583] In some implementations, when a single gRNA molecule is used to target the Cas9 nuclease to generate a double-strand break near the RL2, LAT, or RS1 target, for example, the gRNA is used to target upstream (e.g., within 500 bp upstream) or downstream (e.g., within 500 bp downstream) of the RS1, RL2, or LAT target.

[0584] In some implementations, two or more (e.g., three or four) gRNA molecules are used with one Cas9 molecule or Cas9-fusion protein. In some implementations, when two or more (e.g., three or four) gRNAs are used with two or more Cas9 molecules or Cas9-fusion proteins, at least one Cas9 molecule originates from a different species than the others. When two gRNAs are designed to target two Cas9 molecules, one Cas9 can be from one species, and the second Cas9 can be from a different species. If desired, both Cas9 species are used to generate single-strand or double-strand breaks.

[0585] Any upstream gRNA described in Table 1-12 can pair with any downstream gRNA described in Table 1-12. When an upstream gRNA designed for one Cas9 species pairs with a downstream gRNA designed for Cas9 species of different species, both Cas9 species are used to generate single-strand or double-strand breaks as needed.

[0586] 7. Cas9 molecule

[0587] Cas9 molecules from multiple species can be used in the methods and compositions described in this invention. Although the Cas9 molecules of Streptococcus pyogenes, Staphylococcus aureus, and Neisseria meningitidis are the subject of most of the disclosure of this invention, Cas9 molecules of other species listed herein, derived from, or based on the Cas9 protein, can also be used. These include, for example, Cas9 molecules from the following: *Acidovorax avenae*, *Actinobacillus pleuropneumoniae*, *Actinobacillus succinogenes*, *Actinobacillus suis*, *Actinomyces sp.*, *Cyclophilus denitrificans*, *Aminomonas paucivorans*, *Bacillus cereus*, *Bacillus smithii*, *Bacillus thuringiensis*, *Bacteroides sp.*, *Blastopirellula marina*, and *Bradyrhizobium sp.*), Brevibacillus laterosporus, Campylobacter coli, Campylobacter jejuni, Campylobacter lari, Candidatus puniceispirillum, Clostridium cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphtheria, Corynebacterium matruchotii, Dinoroseobacter shibae, Eubacterium dolichum, gamma proteobacterium, Gluconacetobacter diazotrophic Haemophilus parainfluenzae, Haemophilus sputorum, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae, Ilyobacter polytropus, Kingella kingae, Lactobacillus crispatus, Listeria ivanovii, Listeria monocytogenes, Listeriaceae bacterium, Methylocystis sp., Methylosinus trichosporium, Mobiluncus mulieris, Neisseria bacilliformis, Neisseria cinerea, Neisseria palea (flavescens), Neisseria lactamica, Neisseria sp.Neisseria wadsworthii, Nitrosomonas sp., Parvibaculum lavamentivorans, Pasteurella multocida, Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonas palustris, Rhodovulum sp., Simonsiella muelleri, Sphingomonas sp., Sporolactobacillus vineae, Staphylococcus lugdunensis, Streptococcus sp., Subdoligranulum sp., Tistrella mobilis, Treponema sp.) or Verminephrobacter eiseniae. .

[0588] 7.1 Cas9 Structural Domains

[0589] The crystal structures of two distinct naturally occurring bacterial Cas9 molecules (Jinek 2014) and Streptococcus pyogenes Cas9 with guide RNA (e.g., a synthetic fusion of crRNA and tracrRNA) have been determined (Nishimasu 2014; Anders 2014).

[0590] The naturally occurring Cas9 molecule contains two types of blades: a recognition (REC) blade and a nuclease (NUC) blade; each of which further contains the domains described herein. Figures 8A-8B A schematic diagram of the organization of the primary structure of the important Cas9 domains is provided. The domain nomenclature and amino acid residue numbering used throughout this disclosure are as previously described (Nishimasu 2014). The amino acid residue numbering is based on Cas9 from Streptococcus pyogenes.

[0591] The REC leaflet contains an arginine-rich bridged helix (BH), a REC1 domain, and a REC2 domain. The REC leaflet does not share structural similarity with other known proteins, indicating that it is a Cas9-specific functional domain. The BH domain is a long, helical, arginine-rich region containing amino acids 60-93 of the *Streptococcus pyogenes* Cas9 sequence. The REC1 domain is important for the recognition of repeat:anti-repetitive duplexes, such as gRNA or tracrRNA, and is therefore crucial for Cas9 activity that recognizes target sequences. The REC1 domain contains two REC1 motifs at amino acids 94-179 and 308-717 of the *Streptococcus pyogenes* Cas9 sequence. Although separated by the REC2 domain in the linear primary structure, these two REC1 domains assemble in the tertiary structure to form the REC1 domain. The REC2 domain, or a portion thereof, may also play a role in the recognition of repeat:anti-repetitive duplexes. The REC2 domain includes amino acids 180-307 of the *Streptococcus pyogenes* Cas9 sequence.

[0592] The NUC leaflet contains a RuvC domain, an HNH domain, and a PAM interaction (PI) domain. The RuvC domain shares structural similarity with members of the retroviral integrase superfamily and cleaves single-stranded (e.g., non-complementary) target nucleic acid molecules. The RuvC domain is assembled from three segmented RuvC motifs (RuvCI, RuvCII, and RuvCIII, commonly referred to in the art as the RuvCI domain or the N-terminal RuvC domain, RuvCII domain, and RuvCIII domain) at amino acids 1-59, 718-769, and 909-1098 of the *Streptococcus pyogenes* Cas9 sequence. Similar to the REC1 domain, the three RuvC motifs are linearly separated by other domains in the primary structure. However, in the tertiary structure, these three RuvC motifs assemble to form the RuvC domain. The HNH domain shares structural similarity with HNH endonucleases and cleaves single-stranded (e.g., non-complementary) target nucleic acid molecules. The HNH domain is located between the RuvC II-III motifs and contains amino acids 775-908 of the Streptococcus pyogenes Cas9 sequence. The PI domain interacts with the PAM of the target nucleic acid molecule and contains amino acids 1099-1368 of the Streptococcus pyogenes Cas9 sequence.

[0593] 7.1.1 RuvC-like domains and HNH-like domains

[0594] In some embodiments, the Cas9 molecule or Cas9 peptide comprises an HNH-like domain and a RuvC-like domain, and in some of these embodiments, the cleavage activity depends on the RuvC-like domain and the HNH-like domain. The Cas9 molecule or Cas9 peptide may comprise one or more of the RuvC-like domain and the HNH-like domain. In some embodiments, the Cas9 molecule or Cas9 peptide comprises a RuvC-like domain (e.g., the RuvC-like domain described below) and / or an HNH-like domain (e.g., the HNH-like domain described below).

[0595] RuvC-like structural domain

[0596] In some embodiments, the RuvC-like domain cleaves a single strand (e.g., a non-complementary strand) of the target nucleic acid molecule. A Cas9 molecule or Cas9 polypeptide may include more than one RuvC-like domain (e.g., one, two, three, or more RuvC-like domains). In some embodiments, the RuvC-like domain is at least 5, 6, 7, or 8 amino acids long, but no more than 20, 19, 18, 17, 16, or 15 amino acids long. In some embodiments, the Cas9 molecule or Cas9 polypeptide includes an N-terminal RuvC-like domain of about 10 to 20 amino acids (e.g., about 15 amino acids) in length.

[0597] 7.1.2 N-terminal RuvC-like structural domain

[0598] Some naturally occurring Cas9 molecules contain more than one RuvC-like domain, where cleavage depends on the N-terminal RuvC-like domain. Therefore, Cas9 molecules or Cas9 peptides can contain an N-terminal RuvC-like domain. Exemplary N-terminal RuvC-like domains are described below.

[0599] In some embodiments, the Cas9 molecule or Cas9 polypeptide includes an N-terminal RuvC-like domain comprising an amino acid sequence having formula I:

[0600] D-X1-G-X2-X3-X4-X5-G-X6-X7-X8-X9 (SEQ ID NO: 20),

[0601] in

[0602] X1 is selected from I, V, M, L and T (for example, selected from I, V and L);

[0603] X2 is selected from T, I, V, S, N, Y, E and L (for example, selected from T, V and I);

[0604] X3 is selected from N, S, G, A, D, T, R, M, and F (e.g., A or N);

[0605] X4 is selected from S, Y, N and F (e.g., S);

[0606] X5 is selected from V, I, L, C, T, and F (for example, selected from V, I, and L);

[0607] X6 is selected from W, F, V, Y, S and L (e.g., W);

[0608] X7 is selected from A, S, C, V, and G (for example, selected from A and S);

[0609] X8 is selected from V, I, L, A, M, and H (for example, selected from V, I, M, and L); and

[0610] X9 is selected from any amino acid or is not present (e.g., selected from T, V, I, L, Δ, F, S, A, Y, M and R, or for example, selected from T, V, I, L and Δ).

[0611] In some embodiments, the N-terminal RuvC-like domain differs from the sequence of SEQ ID NO:20 by up to 1 but no more than 2, 3, 4 or 5 residues.

[0612] In some embodiments, the N-terminal RuvC-like structural domain is cleaving-capable. In other embodiments, the N-terminal RuvC-like structural domain is not cleaving-capable.

[0613] In some embodiments, the Cas9 molecule or Cas9 polypeptide includes an N-terminal RuvC-like domain comprising an amino acid sequence having formula II:

[0614] D-X1-G-X2-X3-S-X5-G-X6-X7-X8-X9 (SEQ ID NO: 21),

[0615] in

[0616] X1 is selected from I, V, M, L and T (for example, selected from I, V and L);

[0617] X2 is selected from T, I, V, S, N, Y, E and L (for example, selected from T, V and I);

[0618] X3 is selected from N, S, G, A, D, T, R, M, and F (e.g., A or N);

[0619] X5 is selected from V, I, L, C, T, and F (for example, selected from V, I, and L);

[0620] X6 is selected from W, F, V, Y, S and L (e.g., W);

[0621] X7 is selected from A, S, C, V, and G (for example, selected from A and S);

[0622] X8 is selected from V, I, L, A, M, and H (for example, selected from V, I, M, and L); and

[0623] X9 is selected from any amino acid or is not present (e.g., selected from T, V, I, L, Δ, F, S, A, Y, M and R, or selected from, for example, T, V, I, L and Δ).

[0624] In some embodiments, the N-terminal RuvC-like domain differs from the sequence of SEQ ID NO:21 by up to 1 but no more than 2, 3, 4 or 5 residues.

[0625] In some embodiments, the N-terminal RuvC-like domain comprises an amino acid sequence having Formula III:

[0626] DIG-X2-X3-SVGWA-X8-X9 (SEQ ID NO:22),

[0627] in

[0628] X2 is selected from T, I, V, S, N, Y, E and L (for example, selected from T, V and I);

[0629] X3 is selected from N, S, G, A, D, T, R, M, and F (e.g., A or N);

[0630] X8 is selected from V, I, L, A, M, and H (for example, selected from V, I, M, and L); and

[0631] X9 is selected from any amino acid or is not present (e.g., selected from T, V, I, L, Δ, F, S, A, Y, M and R, or selected from, for example, T, V, I, L and Δ).

[0632] In some embodiments, the N-terminal RuvC-like domain differs from the sequence of SEQ ID NO:22 by up to 1 but no more than 2, 3, 4 or 5 residues.

[0633] In some embodiments, the N-terminal RuvC-like domain comprises an amino acid sequence having formula IV:

[0634] DIGTNSVGWAVX (SEQ ID NO:23),

[0635] in

[0636] X is a nonpolar alkyl amino acid or a hydroxy amino acid, for example, X is selected from V, I, L, and T (e.g., a Cas9 molecule may contain the amino acids shown below). Figure 2A-2G The N-terminal RuvC-like structural domain in the middle (described as Y)).

[0637] In some embodiments, the N-terminal RuvC-like domain differs from the sequence of SEQ ID NO:23 by up to one but no more than two, three, four, or five residues. In some embodiments, the N-terminal RuvC-like domain differs from the sequence of the present invention (e.g., in...). Figures 3A-3B The N-terminal RuvC-like domains disclosed in the Chinese version differ in sequence by up to one but no more than two, three, four, or five residues. In one embodiment, in Figures 3A-3B One, two, three, or all of the highly conserved residues identified in the sample are present.

[0638] In some embodiments, the N-terminal RuvC-like structural domain is related to the present invention (e.g., in...). Figures 4A-4B The N-terminal RuvC-like domains disclosed in the Chinese version differ in sequence by up to one, but no more than two, three, four, or five residues. In some embodiments, in Figures 4A-4B One, two, or all of the highly conserved residues identified in the sample are present.

[0639] 7.1.3 Other RuvC-like structural domains

[0640] In addition to the N-terminal RuvC-like domain, the Cas9 molecule or Cas9 polypeptide may contain one or more additional RuvC-like domains. In some embodiments, the Cas9 molecule or Cas9 polypeptide contains two additional RuvC-like domains. In some embodiments, the additional RuvC-like domains are at least 5 amino acids long, and for example, less than 15 amino acids long, for example, 5 to 10 amino acids long, for example, 8 amino acids long.

[0641] The additional RuvC-like domain can contain an amino acid sequence with the chemical formula V:

[0642] I-X1-X2-E-X3-ARE(SEQ ID NO:15)

[0643] in,

[0644] X1 is V or H;

[0645] X2 is I, L, or V (e.g., I or V); and

[0646] X3 is either M or T.

[0647] In some embodiments, an additional RuvC-like domain comprises an amino acid sequence having the chemical formula VI:

[0648] IV-X2-EMARE (SEQ ID NO:16),

[0649] in

[0650] X2 is I, L, or V (e.g., I or V) (e.g., a Cas9 molecule or Cas9 polypeptide may contain the following shown). Figure 2A-2G Another RuvC-like structural domain (described as B) in the text.

[0651] The additional RuvC-like domain may contain an amino acid sequence with chemical formula VII:

[0652] HHA-X1-DA-X2-X3 (SEQ ID NO:17),

[0653] in

[0654] X1 is H or L;

[0655] X2 is either R or V; and

[0656] X3 is either E or V.

[0657] In some embodiments, the additional RuvC-like domain comprises the following amino acid sequence: HHAHDAYL (SEQ ID NO:18).

[0658] In some implementations, the additional RuvC-like domains differ from the sequences of SEQ ID NO:15-18 by up to one but no more than two, three, four, or five residues.

[0659] In some embodiments, the sequence flanking the N-terminal RuvC-like domain has the amino acid sequence of formula VIII:

[0660] K-X1'-Y-X2'-X3'-X4'-ZTD-X9'-Y(SEQ ID NO:19),

[0661] in

[0662] X1' is selected from K and P;

[0663] X2' is selected from V, L, I and F (e.g., V, I and L);

[0664] X3' is selected from G, A, and S (e.g., G);

[0665] X4' is selected from L, I, V and F (e.g., L);

[0666] X9' is selected from D, E, N, and Q; and

[0667] Z is an N-terminal RuvC-like domain, for example, as described above, having 5 to 20 amino acids.

[0668] 7.1.4 HNH-like structural domain

[0669] In some embodiments, the HNH-like domain cleaves a single-stranded complementary domain (e.g., the complementary strand) of a double-stranded nucleic acid molecule. In some embodiments, the HNH-like domain is at least 15, 20, or 25 amino acids long but no more than 40, 35, or 30 amino acids long, for example, 20 to 35 amino acids long, or 25 to 30 amino acids long. Exemplary HNH-like domains are described below.

[0670] In some embodiments, the Cas9 molecule or Cas9 polypeptide includes an HNH-like domain having an amino acid sequence of formula IX:

[0671] X1-X2-X3-H-X4-X5-P-X6-X7-X8-X 9 -X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -NX 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -N(SEQ ID NO:25), where

[0672] X1 is selected from D, E, Q, and N (e.g., D and E);

[0673] X2 is selected from L, I, R, Q, V, M, and K;

[0674] X3 is selected from D and E;

[0675] X4 is selected from I, V, T, A, and L (e.g., A, I, and V);

[0676] X5 is selected from V, Y, I, L, F and W (e.g., V, I and L);

[0677] X6 is selected from Q, H, R, K, Y, I, L, F, and W;

[0678] X7 is selected from S, A, D, T, and K (e.g., S and A);

[0679] X8 is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g., F);

[0680] X9 is selected from L, R, T, I, V, S, C, Y, K, F and G;

[0681] X 10 Selected from K, Q, Y, T, F, L, W, M, A, E, G, and S;

[0682] X 11 Selected from D, S, N, R, L, and T (e.g., D);

[0683] X 12 Selected from D, N, and S;

[0684] X 13 Selected from S, A, T, G, and R (e.g., S);

[0685] X 14 Selected from I, L, F, S, R, Y, Q, W, D, K, and H (e.g., I, L, and F);

[0686] X 15 Selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y, and V;

[0687] X 16 Selected from K, L, R, M, T, and F (e.g., L, R, and K);

[0688] X 17 Selected from V, L, I, A, and T;

[0689] X 18 Selected from L, I, V, and A (e.g., L and I);

[0690] X 19 Selected from T, V, C, E, S, and A (e.g., T and V);

[0691] X 20 Selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H, and A;

[0692] X 21 Selected from S, P, R, K, N, A, H, Q, G, and L;

[0693] X 22 Selected from D, G, T, N, S, K, A, I, E, L, Q, R, and Y; and

[0694] X 23 Selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D, and F.

[0695] In some embodiments, the HNH-like domain differs from the sequence of SEQ ID NO:25 by at least one but no more than 2, 3, 4, or 5 residues.

[0696] In some embodiments, the HNH-like domains are cleaving-like. In other embodiments, the HNH-like domains are not cleaving-like.

[0697] In some embodiments, the Cas9 molecule or Cas9 polypeptide includes an HNH-like domain comprising an amino acid sequence having the chemical formula X:

[0698] X1-X2-X3-H-X4-X5-P-X6-S-X8-X9-X 10 -DDSX 14 -X 15 -NKVLX 19 -X 20 -X 21 -X 22 -X 23 -N(SEQ ID NO:26),

[0699] in

[0700] X1 is selected from D and E;

[0701] X2 is selected from L, I, R, Q, V, M, and K;

[0702] X3 is selected from D and E;

[0703] X4 is selected from I, V, T, A, and L (e.g., A, I, and V);

[0704] X5 is selected from V, Y, I, L, F and W (e.g., V, I and L);

[0705] X6 is selected from Q, H, R, K, Y, I, L, F, and W;

[0706] X8 is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g., F);

[0707] X9 is selected from L, R, T, I, V, S, C, Y, K, F and G;

[0708] X 10 Selected from K, Q, Y, T, F, L, W, M, A, E, G, and S;

[0709] X 14 Selected from I, L, F, S, R, Y, Q, W, D, K, and H (e.g., I, L, and F);

[0710] X15 Selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y, and V;

[0711] X 19 Selected from T, V, C, E, S, and A (e.g., T and V);

[0712] X 20 Selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H, and A;

[0713] X 21 Selected from S, P, R, K, N, A, H, Q, G, and L;

[0714] X 22 Selected from D, G, T, N, S, K, A, I, E, L, Q, R, and Y; and

[0715] X 23 Selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D, and F.

[0716] In some implementations, the HNH-like domain differs from the sequence of SEQ ID NO:26 by 1, 2, 3, 4, or 5 residues.

[0717] In some embodiments, the Cas9 molecule or Cas9 polypeptide includes an HNH-like domain comprising an amino acid sequence having the chemical formula XI:

[0718] X1-V-X3-HIVP-X6-S-X8-X9-X 10 -DDSX 14 -X 15 -NKVLTX 20 -X 21 -X 22 -X 23 -N(SEQ ID NO:27),

[0719] in

[0720] X1 is selected from D and E;

[0721] X3 is selected from D and E;

[0722] X6 is selected from Q, H, R, K, Y, I, L, and W;

[0723] X8 is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g., F);

[0724] X9 is selected from L, R, T, I, V, S, C, Y, K, F and G;

[0725] X 10 Selected from K, Q, Y, T, F, L, W, M, A, E, G, and S;

[0726] X 14 Selected from I, L, F, S, R, Y, Q, W, D, K, and H (e.g., I, L, and F);

[0727] X 15 Selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y, and V;

[0728] X 20 Selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H, and A;

[0729] X 21 Selected from S, P, R, K, N, A, H, Q, G, and L;

[0730] X 22 Selected from D, G, T, N, S, K, A, I, E, L, Q, R, and Y; and

[0731] X 23 Selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D, and F.

[0732] In some implementations, the HNH-like domain differs from the sequence of SEQ ID NO:27 by 1, 2, 3, 4, or 5 residues.

[0733] In some embodiments, the Cas9 molecule or Cas9 polypeptide includes an HNH-like domain having the amino acid sequence of formula XII:

[0734] D-X2-DHI-X5-PQ-X7-F-X9-X 10 -DX 12 -SIDNX 16 -VLX 19 -X 20 -SX 22 -X 23 -N(SEQID NO:28),

[0735] in

[0736] X2 is selected from I and V;

[0737] X5 is selected from I and V;

[0738] X7 is selected from A and S;

[0739] X9 is selected from I and L;

[0740] X 10 Selected from K and T;

[0741] X 12 Selected from D and N;

[0742] X 16 Selected from R, K, and L;

[0743] X 19 Selected from T and V;

[0744] X 20 Selected from S and R;

[0745] X 22 Selected from K, D, and A; and

[0746] X 23 Selected from E, K, G, and N (e.g., Cas9 molecules or Cas9 peptides may contain HNH-like domains as described herein).

[0747] In some embodiments, the HNH-like domain differs from the sequence of SEQ ID NO:28 by up to 1 but no more than 2, 3, 4 or 5 residues.

[0748] In some embodiments, the Cas9 molecule or Cas9 polypeptide comprises an amino acid sequence having the chemical formula XIII:

[0749] LYYLQNG-X1'-DMY-X2'-X3'-X4'-X5'-LDI-X6'-X7'-LS-X8'-YZNR-X9'-KX 10 '-DX 11 '-VP(SEQ ID NO:24),

[0750] in

[0751] X1' is selected from K and R;

[0752] X2' is selected from V and T;

[0753] X3' is selected from G and D;

[0754] X4' is selected from E, Q, and D;

[0755] X5' is selected from E and D;

[0756] X6' is selected from D, N, and H;

[0757] X7' is selected from Y, R, and N;

[0758] X8' is selected from Q, D, and N;

[0759] X9' is selected from G and E;

[0760] X 10 Selected from S and G;

[0761] X 11 'Selected from D and N; and

[0762] Z is an HNH-like structural domain, such as those described above.

[0763] In some embodiments, the Cas9 molecule or Cas9 polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO:24 by up to one but no more than two, three, four, or five residues.

[0764] In some implementations, the HNH-like domain is similar to that described herein (e.g., in...). Figures 5A-5C The sequences of the HNH-like domains disclosed in the (Chinese) specification differ by as many as one, but no more than two, three, four, or five residues. In some embodiments, in Figures 5A-5C One or two of the highly conserved residues identified in the sample are present.

[0765] In some embodiments, the HNH-like structural domain is related to the present invention (e.g., in...). Figures 6A-6B The sequences of the HNH-like domains disclosed in the (Chinese) specification differ by as many as one, but no more than two, three, four, or five residues. In some embodiments, in Figures 6A-6B One, two, three, or all of the highly conserved residues identified in the sample are present.

[0766] 7.2 Cas9 Activity

[0767] In some implementations, Cas9 molecules or Cas9 peptides are capable of cleaving target nucleic acid molecules. Typically, wild-type Cas9 molecules cleave both strands of the target nucleic acid molecule. Cas9 molecules and Cas9 peptides can be engineered to alter nuclease cleavage (or other properties), for example, to provide Cas9 molecules or Cas9 peptides that function as cleaving enzymes or lack the ability to cleave target nucleic acids. Cas9 molecules or Cas9 peptides capable of cleaving target nucleic acid molecules are referred to herein as eaCas9 (enzymatically active Cas9) molecules or eaCas9 peptides.

[0768] In some embodiments, the eaCas9 molecule or eaCas9 polypeptide contains one or more of the following enzymatic activities:

[0769] Cleavage enzyme activity is the ability to cleave single strands (e.g., non-complementary or complementary strands) of nucleic acid molecules.

[0770] Double-stranded nuclease activity is the ability to cleave the two strands of a double-stranded nucleic acid and produce double-strand breaks. In some embodiments, it occurs in the presence of two cleavage enzyme activities.

[0771] Endonuclease activity;

[0772] Exonuclease activity; and

[0773] Helicase activity is the ability of a double-stranded nucleic acid to unwind its helical structure.

[0774] In some embodiments, an enzymatically active Cas9 (“eaCas9”) molecule or eaCas9 polypeptide cleaves two DNA strands, resulting in a double-strand break. In some embodiments, the eaCas9 molecule or eaCas9 polypeptide cleaves only one strand, for example, the strand to which gRNA h...

Claims

1. A genome editing system, the system comprising: (i) a gRNA molecule, or a polynucleotide encoding said gRNA molecule, said gRNA molecule comprising a targeting domain complementary to a target sequence of the herpes simplex virus (HSV) RS1 gene, said targeting domain comprising nucleotide sequences selected from the group consisting of: SEQ ID NO: 243, 2515, 2522, 3362 and 3363; and (ii) Cas9 molecule, or polynucleotide encoding said Cas9 molecule.

2. The genome editing system of claim 1, wherein the target domain is configured to form double-strand breaks or single-strand breaks at HSV target sites of about 500 bp, about 450 bp, about 400 bp, about 350 bp, about 300 bp, about 250 bp, about 200 bp, about 150 bp, about 100 bp, about 50 bp, about 25 bp, or about 10 bp, thereby altering the HSV RS1 gene.

3. The genome editing system of claim 2, wherein the alteration of the HSV RS1 gene comprises knocking out the HSV RS1 gene or knocking down the HSV RS1 gene.

4. The genome editing system according to any one of claims 1-3, wherein the Cas9 molecule is a Staphylococcus aureus Cas9 molecule.

5. The genome editing system according to claim 4, wherein the Staphylococcus aureus Cas9 molecule recognizes any one of the PAMs NNGRRT or NNGRRV.

6. The genome editing system according to any one of claims 1-3, wherein the Cas9 molecule is selected from the group consisting of enzyme-activated Cas9 molecules, enzyme-inactivated Cas9 molecules, and enzyme-inactivated Cas9 fusion proteins.

7. The genome editing system of claim 6, wherein the enzyme activates the Cas9 molecule to include HNH-like domain cleavage activity, but does not have or does not significantly have N-terminal RuvC-like domain cleavage activity.

8. The genome editing system according to claim 6, wherein the enzyme activating the Cas9 molecule is an HNH-like domain nickase.

9. The genome editing system of claim 6, wherein the enzyme-activated Cas9 molecule includes N-terminal RuvC-like domain cleavage activity, but does not have or does not significantly have HNH-like domain cleavage activity.

10. The genome editing system of claim 6, wherein the enzyme activating the Cas9 molecule is an N-terminal RuvC-like domain nickase.

11. The genome editing system according to any one of claims 1-3, wherein the Cas9 molecule comprises a wild-type Cas9 molecule, a mutant Cas9 molecule, or a combination of both.

12. The genome editing system of claim 11, wherein the mutant Cas9 molecule comprises a mutation selected from the group consisting of D10, E762, D986, H840, N854, N863 and N580.

13. The genome editing system according to any one of claims 1-3, wherein the gRNA is a modular gRNA molecule or a chimeric gRNA molecule.

14. The genome editing system according to any one of claims 1-3, wherein the gRNA molecule comprises, from 5' to 3': Targeted structural domain; First complementary structural domain; Connect structural domains; Second complementary structural domain; Proximal structural domain; and Tail structural domain.

15. The genome editing system of claim 14, wherein the length of the linker domain does not exceed 25 nucleotides.

16. The genome editing system of claim 14, wherein the proximal domain and the tail domain together have a length of at least 20, at least 25, at least 30, or at least 40 nucleotides.

17. The genome editing system according to any one of claims 1-3, wherein the system comprises two, three, or four gRNA molecules.

18. The genome editing system according to any one of claims 1-3, for altering the HSVRS1 gene in a cell.

19. The genome editing system of claim 18, wherein the cells are infected with HSV.

20. A composition comprising a gRNA molecule or a polynucleotide encoding the gRNA molecule, said gRNA molecule comprising a targeting domain complementary to a target sequence of the HSV RS1 gene, said targeting domain comprising a nucleotide sequence selected from the group consisting of: SEQ ID NO: 243, 2515, 2522, 3362 and 3363.

21. The composition of claim 20, wherein the composition comprises one, two, three or four gRNA molecules.

22. The composition of claim 20, further comprising at least one Cas9 molecule, or a polynucleotide encoding at least one Cas9 molecule.

23. The composition of claim 22, wherein the at least one Cas9 molecule is a Staphylococcus aureus Cas9 molecule.

24. The composition according to claim 22 or 23, wherein the at least one Cas9 molecule comprises a wild-type Cas9 molecule, a mutant Cas9 molecule, or a combination of both.

25. The composition of claim 24, wherein the mutant Cas9 molecule comprises a mutation selected from the group consisting of D10, E762, D986, H840, N854, N863 and N580.

26. The composition of claim 23, wherein the Staphylococcus aureus Cas9 molecule recognizes any one of the PAMs NNGRRT or NNGRRV.

27. A vector comprising a polynucleotide encoding a gRNA molecule, the gRNA molecule comprising a targeting domain complementary to a target sequence of the HSV RS1 gene, wherein the targeting domain comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 243, 2515, 2522, 3362 and 3363.

28. The vector of claim 27, wherein the vector further comprises a polynucleotide encoding at least one Cas9 molecule.

29. The carrier according to claim 28, wherein the at least one Cas9 molecule is a Staphylococcus aureus Cas9 molecule.

30. The vector according to claim 28 or 29, wherein the at least one Cas9 molecule comprises a wild-type Cas9 molecule, a mutant Cas9 molecule, or a combination of both.

31. The vector of claim 30, wherein the mutant Cas9 molecule comprises a mutation selected from the group consisting of D10, E762, D986, H840, N854, N863 and N580.

32. The vector according to claim 29, wherein the Staphylococcus aureus Cas9 molecule recognizes any one of the PAMs NNGRRT or NNGRRV.

33. The vector according to any one of claims 27-29, wherein the vector is a viral vector.

34. An in vitro method for altering the HSV RS1 gene in cells, the method comprising administering to the cells one of the following: (i) A gene editing system comprising a gRNA molecule and at least one Cas9 molecule, the gRNA molecule comprising a targeting domain complementary to a targeting sequence of the HSV RS1 gene; (ii) A vector comprising a polynucleotide encoding a gRNA molecule and a polynucleotide encoding a Cas9 molecule, wherein the gRNA molecule comprises a targeting domain complementary to the targeting sequence of the HSV RS1 gene; or (iii) A composition comprising a gRNA molecule and at least one Cas9 molecule, the gRNA molecule comprising a targeting domain complementary to the targeting sequence of the HSV RS1 gene. The targeting domain is composed of nucleotide sequences selected from the following group: SEQ ID NO: 243, 2515, 2522, 3362 and 3363.

35. The in vitro method of claim 34, wherein the alteration comprises knocking out or knocking down the HSV RS1 gene.

36. The in vitro method according to claim 34 or 35, wherein the cells are infected with HSV.

37. The in vitro method according to claim 34 or 35, wherein the Cas9 molecule is a Staphylococcus aureus Cas9 molecule.

38. A gRNA molecule comprising a targeting domain complementary to a target sequence of an intracellular HSV RS1 gene, wherein the targeting domain comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 243, 2515, 2522, 3362 and 3363.

39. A cell comprising a genome editing system according to any one of claims 1-3, a composition according to any one of claims 20-23, or a vector according to any one of claims 27-29, wherein the cell is selected from the group consisting of epithelial cells, nerve cells, and optic nerve cells.

40. Use of one or more of the following in the preparation of a medicament for altering the HSV RS1 gene in cells: (i) A gene editing system comprising a gRNA molecule and at least one Cas9 molecule, the gRNA molecule comprising a targeting domain complementary to a targeting sequence of the HSV RS1 gene; (ii) A vector comprising a polynucleotide encoding a gRNA molecule and a polynucleotide encoding a Cas9 molecule, wherein the gRNA molecule comprises a targeting domain complementary to the targeting sequence of the HSV RS1 gene; or (iii) A composition comprising a gRNA molecule and at least one Cas9 molecule, the gRNA molecule comprising a targeting domain complementary to the targeting sequence of the HSV RS1 gene. The targeting domain is composed of nucleotide sequences selected from the following group: SEQ ID NO: 243, 2515, 2522, 3362 and 3363.

41. The use according to claim 40, wherein the alteration includes knocking out or knocking down the HSV RS1 gene.

42. The use according to claim 40 or 41, wherein the cells are infected with HSV.

43. The use according to claim 40 or 41, wherein the Cas9 molecule is a Staphylococcus aureus Cas9 molecule.

Images