Novel crispr enzymes and systems

Abstract

Embodiments disclosed herein are directed to engineered CRISPR-Cas effector proteins that comprise at least one modification compared to an unmodified CRISPR-Cas effector protein that enhances binding of the of the CRISPR complex to the binding site and/or alters editing preference as compared to wild type. In certain example embodiments, the CRISPR-Cas effector protein is a Type V effector protein. In certain other example embodiments, the Type V effector protein is Cpf1. Embodiments disclosed herein are directed to viral vectors for delivery of CRISPR-Cas effector proteins, including Cpf1. In certain example embodiments, the vectors are designed so as to allow packaging of the CRISPR-Cas effector protein within a single vector. There is also an increased interest in the design of compact promoters for packing and thus expressing larger transgenes for targeted delivery and tissue-specificity. Thus, in another aspect certain embodiments disclosed herein are directed to delivery vectors, constructs, and methods of delivering larger genes for systemic delivery.

1 . An engineered CRISPR-Cas effector protein, wherein the protein complexes with a nucleic acid molecule comprising a guide sequence to form a CRISPR complex, wherein in the CRISPR complex the nucleic acid molecule targets one or more polynucleotide loci and the protein comprises at least one modification compared to the unmodified protein that enhances binding of the CRISPR complex to the binding site and/or alters editing preference as compared to wild type. 
     
     
         2 . The engineered CRISPR-Cas effector protein of  claim 1 , wherein the editing preference is for indel formation, or
 wherein the at least one modification increases formation of one or more specific indels, preferably, wherein the CRISPR-Cas effector protein is a Class V CRISPR-Cas effector protein, preferably, wherein the Class V CRISPR-Cas effector protein is Cpf1 or an orthologue thereof,   optionally, wherein the at least one modification ins in a C-terminal RuvC like domain, a N-terminal alpha-helical region, a mixed alpha and beta region, or a combination thereof, optionally, wherein the at least one modification results in insertion of an A adjacent to an A, T, G, or C in a target region, insertion of a T adjacent to an A, T, G, or C in the target region, insertion of a G adjacent to an A, T, G, or C, insertion of a C adjacent to an A, T, C, or G, or a combination thereof.   
     
     
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         8 . The engineered CRISPR-Cas effector protein of  claim 1 , further comprising at least one additional mutation that alters the binding property of the effector protein as to the nucleic acid molecule comprising the guide sequence or the target polynucleotide loci, alters binding kinetics as to the nucleic acid molecule or target polynucleotide or alters binding specificity as to the nucleic acid molecule. 
     
     
         9 . An engineered system for modifying a target locus of interest comprising;
 (a) a guide molecule which comprises a guide sequence, or a nucleotide encoding a guide molecule; and   (b) the CRISPR-Cas effector of  claim 1 , or a nucleotide encoding said CRISPR-Cas effector protein,   optionally, wherein the components (a) and (b) are encoded on the same or different vectors.   
     
     
         10 . (canceled) 
     
     
         11 . A method for developing or designing a CRISPR-Cas system-based therapy or therapeutic, comprising:
 optionally, selecting one or more therapeutic targets,   optionally, selecting one or more CRISPR-Cas system functionalities,   optionally, selecting one or more CRISPR-Cas system mode of delivery,   optionally, selecting one or more CRISPR-Cas system delivery vehicle or expression system, and   optimization of selected parameters or variables associated with the CRISPR-Cas system and/or its functionality,   wherein specificity, efficacy, and/or safety are optimized.   
     
     
         12 . The method according to  claim 11 , wherein the selected parameters or variables are selected from the group comprising CRISPR effector specificity, gRNA specificity, CRISPR-Cas complex specificity, PAM restrictiveness, PAM type (natural or modified), PAM nucleotide content, PAM length, CRISPR effector activity, gRNA activity, CRISPR-Cas complex activity, target cleavage efficiency, target site selection, target sequence length, ability of effector protein to access regions of high chromatin accessibility, degree of uniform enzyme activity across genomic targets, epigenetic tolerance, mismatch/budge tolerance, CRISPR effector stability, CRISPR effector mRNA stability, gRNA stability, CRISPR-Cas complex stability, CRISPR effector protein or mRNA immunogenicity or toxicity, gRNA immunogenicity or toxicity, CRISPR-Cas complex immunogenicity or toxicity, CRISPR effector protein or mRNA dose or titer, gRNA dose or titer, CRISPR-Cas complex dose or titer, CRISPR effector protein size, CRISPR effector expression level, gRNA expression level, CRISPR-Cas complex expression level, CRISPR effector spatiotemporal expression, gRNA spatiotemporal expression, CRISPR-Cas complex spatiotemporal expression. 
     
     
         13 . The method according to  claim 11 ,
 wherein optimization of specificity comprises optimizing one or more parameters or variables selected from CRISPR effector specificity, gRNA specificity, CRISPR-Cas complex specificity, PAM restrictiveness, PAM type (natural or modified), PAM nucleotide content, PAM length,   wherein optimization of efficacy comprises optimizing one or more parameters or variables selected from CRISPR effector activity, gRNA activity, CRISPR-Cas complex activity, target cleavage efficiency, target site selection, target sequence length, CRISPR effector protein size, ability of effector protein to access regions of high chromatin accessibility, degree of uniform enzyme activity across genomic targets, epigenetic tolerance, mismatch/budge tolerance, and   wherein optimization of safety comprises optimizing one or more parameters or variables selected from CRISPR effector stability, CRISPR effector mRNA stability, gRNA stability, CRISPR-Cas complex stability, CRISPR effector protein or mRNA immunogenicity or toxicity, gRNA immunogenicity or toxicity, CRISPR-Cas complex immunogenicity or toxicity, CRISPR effector protein or mRNA dose or titer, gRNA dose or titer, CRISPR-Cas complex dose or titer, CRISPR effector expression level, gRNA expression level, CRISPR-Cas complex expression level, CRISPR effector spatiotemporal expression, gRNA spatiotemporal expression, CRISPR-Cas complex spatiotemporal expression;   optionally, wherein CRISPR effector specificity is optimized by selecting the most specific CRISPR effector, such as by selecting the most specific CRISPR effector orthologue or by specific CRISPR effector mutations which increase specificity,   gRNA specificity is optimized by selecting the most specific gRNA, such as by selecting gRNA having low homology, i.e. at least one or preferably more, such as at least 2, or preferably at least 3, mismatches to off-target sites,   PAM restrictiveness is optimized by selecting a CRISPR effector having to most restrictive PAM recognition, such as by selecting a CRISPR effector orthologue having more restrictive PAM recognition or by specific CRISPR effector mutations which increase or alter PAM restrictiveness,   CRISPR effector activity is optimized by selecting the most active CRISPR effector, such as by selecting the most active CRISPR effector orthologue or by specific CRISPR effector mutations which increase activity,   gRNA activity is optimized by selecting the most active gRNA such as by increasing gRNA stability through RNA modification,   target site selection is optimized by selecting the optimal position of the target site within a gene, locus or other genomic region, such as by selecting a target site in an early and/or conserved exon or domain having low variability, such as polymorphisms, within a population, or by minimization of off-target effects, such as off-targets qualified as having 1-5, 1-4, or preferably 1-3 mismatches compared to target, preferably also taking into account variability within a population,   CRISPR effector stability is optimized by selecting CRISPR effector having appropriate half-life, such as preferably a short half-life while still capable of maintaining sufficient activity, such as by selecting an appropriate CRISPR effector orthologue having a specific half-life or by specific CRISPR effector mutations or modifications which affect half-life or stability, such as inclusion of stabilizing or destabilizing domains or sequences,   CRISPR effector mRNA stability is optimized by increasing or decreasing CRISPR effector mRNA stability, such as by increasing or decreasing CRISPR effector mRNA stability through mRNA modification,   gRNA stability is optimized by increasing or decreasing gRNA stability, such as by increasing or decreasing gRNA stability through RNA modification,   CRISPR effector protein or mRNA immunogenicity or toxicity is optimized by decreasing CRISPR effector protein or mRNA immunogenicity or toxicity, such as by mRNA or protein modifications,   gRNA immunogenicity or toxicity is optimized by decreasing gRNA immunogenicity or toxicity, such as by gRNA modifications,   CRISPR effector protein or mRNA dose or titer is optimized by selecting dosage or titer to minimize toxicity and/or maximize specificity and/or efficacy,   gRNA dose or titer is optimized by selecting dosage or titer to minimize toxicity and/or maximize specificity and/or efficacy,   CRISPR effector protein size is optimized by selecting minimal protein size to increase efficiency of delivery, in particular for virus mediated delivery,   CRISPR effector, gRNA, and/or CRISPR-Cas complex expression level is optimized by limiting or extending the duration of expression and/or limiting or increasing expression level, such as by using self-inactivating CRISPR-Cas systems, such as including a self-targeting gRNA, by using viral vectors having limited expression duration, by using appropriate promoters for low or high expression levels, by combining different delivery methods for individual CRISP-Cas system components, such as virus mediated delivery of CRISPR-effector encoding nucleic acid combined with non-virus mediated delivery of gRNA, or virus mediated delivery of gRNA combined with non-virus mediated delivery of CRISPR effector protein or mRNA, and   CRISPR effector, gRNA, or CRISPR-Cas complex spatiotemporal expression is optimized by appropriate choice of conditional and/or inducible expression systems, including controllable CRISPR effector activity optionally a destabilized CRISPR effector and/or a split CRISPR effector, and/or cell- or tissue-specific expression systems;   optionally, wherein gRNA specificity is optimized at the population level of the target organism; and   optionally, wherein optimization of gRNA specificity comprises minimizing gRNA target site sequence variation across a population and/or minimizing gRNA off-target incidence across a population.   
     
     
         14 . The method according to  claim 11 , wherein optimization of selected parameters or variables associated with the CRISPR-Cas system and/or its functionality depends on the therapeutic target or therapeutic targets, the mode or type of CRISPR-Cas system based therapeutic target(s) modulation, modification, or manipulation, and/or the delivery of the CRISPR-Cas system components;
 optionally, wherein optimization of selected parameters or variables associated with the CRISPR-Cas system and/or its functionality depends on the choice of the therapeutic target, the CRISPR-Cas system functionality, the CRISPR-Cas system mode of delivery, and/or the CRISPR-Cas system delivery vehicle or expression system.   
     
     
         15 . The method according to  claim 11 , wherein the therapeutic target is a single gene, locus, or other genomic site, or multiple genes, loci or other genomic sites. 
     
     
         16 . The method according to  claim 11 , wherein the CRISPR-Cas system based therapy or therapeutics involve
 target disruption, such as target mutation, such as leading to gene knockout,   replacement of particular target sites, such as leading to target correction,   removal of particular target sites, such as leading to target deletion, and/or   modulation of target site functionality, such as target site activity or accessibility, optionally leading to (transcriptional and/or epigenetic) gene or genomic region activation or gene or genomic region silencing.   
     
     
         17 . The method according to  claim 11 , wherein the CRISPR-Cas system functionality comprises
 genomic mutation, such as single genomic mutation or multiple genomic mutation,   gene knockout, such as single gene knockout or multiple gene knockout,   gene correction, such as single gene correction or multiple gene correction,   genomic region deletion, such as single genomic region deletion of multiple genomic region deletion, and/or   gene or genomic region functionality, such as single or multiple gene or genomic region activity.   
     
     
         18 . The method according to  claim 11 , wherein the mode of delivery comprises
 delivering gRNA and/or CRISPR effector protein,   delivering gRNA and/or CRISPR effector mRNA, or   delivering gRNA and/or CRISPR effector as a DNA based expression system.   
     
     
         19 . The method according to  claim 11 , wherein the delivery vehicle and/or expression system comprises liposomes, lipid particles, nanoparticles, biolistics, or viral-based expression/delivery systems, optionally adenoviral, AAV, or lentiviral expression/delivery systems. 
     
     
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         24 . The method according to  claim 13 , comprising
 (a) selecting for a therapeutic locus of interest gRNA target sites, wherein said target sites have minimal sequence variation across a population, and
 from said selected target sites (sub)selecting target sites, wherein a gRNA directed against said target sites recognizes a minimal number of off-target sites across said population, 
   or   (b) selecting for a therapeutic locus of interest gRNA target sites, wherein said target sites have minimal sequence variation across a population, or
 selecting for a therapeutic locus of interest gRNA target sites, wherein a gRNA directed against said target sites recognizes a minimal number of off-target sites across said population, and 
   optionally estimating the number of (sub)selected target sites needed to treat a population,   optionally validating one or more of the (sub)selected target sites for an individual subject,   optionally designing one or more gRNA recognizing one or more of said (sub)selected target sites.   
     
     
         25 . A method for developing or designing a CRISPR-Cas system based therapy or therapeutic or for developing or designing a gRNA for use in a CRISPR-Cas system based therapy or therapeutic, comprising
 (a) selecting, for a therapeutic locus of interest, gRNA target sites, wherein said target sites have minimal sequence variation across a population of a target organism, and
 (sub)selecting one or more target sites from said selected target sites, wherein a gRNA directed against said target sites recognizes a minimal number of off-target sites across said population, or 
   (b) selecting, for a therapeutic locus of interest, gRNA target sites, wherein said target sites have minimal sequence variation across a population of a target organism, or
 selecting, for a therapeutic locus of interest, gRNA target sites, wherein a gRNA directed against said target sites recognizes a minimal number of off-target sites across said population, 
   and
 optionally estimating the number of (sub)selected target sites needed to treat a population, 
 optionally validating one or more of the (sub)selected target sites for an individual subject, 
 optionally designing one or more gRNA recognizing one or more of said (sub)selected target sites; 
   optionally, wherein said method is a method for developing or designing a CRISPR-Cas system based therapy or therapeutic or for developing or designing a gRNA for use in a CRISPR-Cas system based therapy or therapeutic in a population of a target organism.   
     
     
         26 . (canceled) 
     
     
         27 . The method according to  claim 13 , wherein said target sites having minimal sequence variation across a population are characterized by absence of sequence variation in at least 99%, preferably at least 99.9%, more preferably at least 99.99% of the population. 
     
     
         28 . The method according to  claim 13 , wherein said population comprises at least 1000 individuals, such as at least 5000 individuals, such as at least 10000 individuals, such as at least 50000 individuals. 
     
     
         29 . The method according to  claim 13 , wherein said off-target sites are characterized by at least one mismatch between the off-target site and the gRNA, and/or the off-target sites are characterized by at most five, preferably at most four, more preferably at most three mismatches between the off-target site and the gRNA, preferably both. 
     
     
         30 . The method according to  claim 13 , wherein said minimal number of off-target sites across said population is determined for high-frequency haplotypes in said population, optionally, wherein the high-frequency haplotypes are characterized by occurrence in at least 0.1% of the population. 
     
     
         31 . (canceled) 
     
     
         32 . The method according to  claim 24 , wherein the number of (sub)selected variation, such as low frequency sequence variation captured in large scale sequencing datasets,
 optionally, wherein the number of (sub)selected target sites needed to treat a population of a given size is estimated, or   optionally, wherein the (sub)selected target is validated by genome sequencing, preferably whole genome sequencing.   
     
     
         33 . (canceled) 
     
     
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         35 . A method for developing or designing a CRISPR-Cas system based therapy or therapeutic, comprising:
 selecting a set of target sequences for one or more loci in a target population, wherein the target sequences do not contain variants occurring above a threshold allele frequency in the target population;   removing any target sequences having high frequency off-target candidates (relative to other platinum targets in the set) to define a final target sequence set;   preparing a set of CRISPR-Cas systems based on the final target sequence set, wherein a number of CRISP-Cas systems prepared is based at least in part a size of a target population.   
     
     
         36 . The method of  claim 35 , further comprising;
 obtaining genome sequencing data of a subject to be treated; and   treating the subject with a CRISPR-Cas system selected from the set of CRISPR-Cas systems, wherein the CRISPR-Cas system selected is based at least in part on the genome sequencing data of the individual,   optionally, wherein the genome sequencing data is whole genome sequencing data.   
     
     
         37 . (canceled) 
     
     
         38 . The method of  claim 35 , wherein target sequences are further selected based on optimization of one or more parameters consisting of; PAM type (natural or modified), PAM nucleotide content, PAM length, target sequence length, PAM restrictiveness, target cleavage efficiency, and target sequence position within a gene, a locus or other genomic region. 
     
     
         39 . The method of  claim 35 , wherein the effector protein for each CRISPR-Cas system in the set of CRISPR-Cas systems is selected based on optimization of one or more parameters selected from the group consisting of; effector protein size, ability of effector protein to access regions of high chromatin accessibility, degree of uniform enzyme activity across genomic targets, epigenetic tolerance, mismatch/budge tolerance, effector protein specificity, effector protein stability or half-life, effector protein immunogenicity or toxicity 
     
     
         40 . The method of  claim 35 , wherein the guide RNA is a tru guide, an escorted guide, or a protected guide. 
     
     
         41 . The method of  claim 35 , wherein the CRISPR-Cas system functionality comprises genomic mutation, gene knockout, gene correction, genomic region deletion, modulation of gene or genomic region functionality,
 optionally, wherein modulation of gene or genomic region functionality comprising modulation gene activity or accessibility optionally leading to transcriptional and/or epigenetic gene or genomic region activation or gene or genomic region silencing.   
     
     
         42 . (canceled) 
     
     
         43 . The method of  claim 35 , wherein delivery comprises delivering gRNA and/or CRISPR effector protein, delivering gRNA and/or CRISPR effector mRNA, or delivering gRNA and/or CRISPR effector as a DNA based expression system,
 optionally, wherein the delivery vehicle and/or expression system for delivering the CRISPR-Cas systems or components thereof comprises liposomes, lipid particles, nanoparticles, biolistics, or viral-based expression/delivery systems.   
     
     
         44 . (canceled) 
     
     
         45 . The method of  claim 35 , wherein off-target candidates, PAM restrictiveness, target cleavage efficiency, or effector protein specificity is determined using a sequencing-based double-strand break detection assay. 
     
     
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         67 . A eukaryotic cell comprising the system of  claim 46 , preferably wherein the cell is an in vitro, ex vivo or in vivo host cell or cell line or progeny thereof, wherein the host cell or cell line is not a human germ cell line,
 optionally, wherein the cell comprises a stem cell or stem cell line,   optionally, wherein the cell is an animal or plant cell, preferably a human cell.   
     
     
         68 . (canceled) 
     
     
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         70 . A method of modulating gene expression or modifying a target locus of interest in a cell or cell line, wherein the method comprises introducing the system of  claim 46  into the cell or cell line, thereby obtaining a modified cell or cell line, preferably, wherein the modified cell or cell line is further cultured to produce progeny. 
     
     
         71 . (canceled) 
     
     
         72 . A method of modifying a target locus of interest in vitro or ex vivo, the method comprising delivering to said locus a non-naturally occurring or engineered composition comprising the system of  claim 46 , wherein the Cpf1 protein forms a complex with the one or more nucleic acid components and upon binding of the complex to a target locus of interest, the Cpf1 protein induces a modification of the target locus of interest. 
     
     
         73 . A method of producing a plant having a modified trait of interest encoded by a gene of interest, said method comprising contacting a plant cell with a system according to  claim 46 , thereby either modifying or introducing said gene of interest, and regenerating a plant from said plant cell. 
     
     
         74 . A method of modifying an organism by manipulation of one or more target sequences at genomic loci of interest, comprising delivering to the organism the system according to  claim 46  thereby obtaining a modified organism, preferably, wherein the organism is a plant or algae. 
     
     
         75 . (canceled) 
     
     
         76 . An ex vivo method of modifying a cell or cell line by manipulation of one or more target sequences at genomic loci of interest comprising delivering to the cell the system according to  claim 46  thereby obtaining a modified cell or cell line, wherein the method does not comprise a process for modifying the germ line genetic identity of a human being, preferably, wherein the modified cell or cell line is further cultured to produce progeny. 
     
     
         77 . (canceled) 
     
     
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         79 . The system according to  claim 46  for use in therapy,
 preferably, wherein said therapy is for gene or genome editing, or gene therapy; 
 preferably, wherein said therapy is for the treatment of one or more of the following: blood and coagulation diseases and disorders: cell dysregulation and oncology diseases and disorders; inflammation and immune related diseases and disorders: metabolic, liver, kidney and protein diseases and disorders: muscular/skeletal diseases and disorders: neurological and neuronal diseases and disorders; and ocular diseases and disorders. 
 
     
     
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         82 . A cell or cell line obtained by or obtainable by the method of  claim 70 . 
     
     
         83 . A method for developing or designing a CRISPR-Cas system-based therapy or therapeutic, comprising:
 (a) selecting one or more target loci   (b) selecting one or more CRISPR-Cas system functionalities   (c) optionally, selecting one or more modes of delivery   (d) preparing a CRISPR-Cas system selected based on steps (a)-(c).   
     
     
         84 . The method of  claim 83 , wherein selecting one or more target, target sequence, or target loci comprises optimizing one or more of target, target sequence, or target loci location, length, specificity, and PAM characteristics,
 preferably, wherein optimizing target location comprises selecting a target sequence with a gene, locus, or other genomic region having low variability, preferably, wherein low variability comprises selecting an early and/or conserved exon or domain having low variability;   optionally, wherein optimizing target location comprises selecting target loci having an absence of sequence variation in at least 99%, of a population, preferably, wherein the population comprises at least 1000 individuals;   optionally, wherein optimizing target length comprises selecting a target sequence within the one or more target loci between 5 and 25 nucleotides, preferably, wherein target sequence length is 20 nucleotides;   optionally, wherein optimizing target specificity comprises selecting target loci that minimize off-target candidates, preferably, wherein off-target candidates have 1-3 mismatches or distal PAM mismatches, preferably, wherein off-target candidates are identified using a sequencing-based double-strand break (DSB) detection assay, preferably, wherein the sequencing-based DSB detection assay comprises labeling a site of a DSB with an adapter comprising a primer binding site, labeling a site of a DSB with a barcode or unique molecular identifier, or combination thereof.   
     
     
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         95 . The method of  claim 83 , wherein optimizing PAM characteristics comprises optimizing nucleotide content of a PAM, preferably, wherein optimizing nucleotide content of PAM is selecting a PAM with a motif that maximizes abundance in the one or more target loci, minimizes mutation frequency, or both. 
     
     
         96 . (canceled) 
     
     
         97 . The method of  claim 83 , wherein selecting one or more CRISPR-Cas system functionalities comprises selecting one or more of an optimal effector protein, an optimal guide RNA, or both,
 preferably, wherein selecting an optimal effector protein comprises optimizing one or more of effector protein type, size, PAM specificity, effector protein stability, immunogenicity or toxicity, functional specificity, and efficacy, preferably, wherein optimizing size comprises selecting a protein effector having a minimal size;   preferably, wherein the effector protein is a naturally occurring or modified effector protein;   preferably, wherein the modified effector protein is a nickase, a deaminase, or a deactivated effector protein.   
     
     
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         102 . The method of  claim 97 , wherein optimizing a PAM specificity comprises selecting an effector protein having a modified PAM specificity; or wherein optimizing effector protein stability comprises selecting an effector protein having a short half-life while maintaining sufficient activity, such as by selecting an appropriate CRISPR effector orthologue having a specific half-life or stability; or wherein optimizing immunogenicity or toxicity comprises minimizing effector protein immunogenicity or toxicity by protein modifications; or wherein optimizing functional specific comprises selecting a protein effector with reduced tolerance of mismatches and/or bulges between the guide RNA and one or more target loci; or wherein optimizing efficacy comprises optimizing overall efficiency, epigenetic tolerance, or both; or wherein optimizing epigenetic tolerance comprises optimizing methylation tolerance, epigenetic mark competition, or both; or wherein optimizing methylation tolerance comprises selecting an effector protein that modify methylated DNA; or wherein optimizing epigenetic tolerance comprises selecting an effector protein unable to modify silenced regions of a chromosome, selecting an effector protein able to modify silenced regions of a chromosome, or selecting target loci not enriched for epigenetic markers,
 preferably, wherein maximizing overall efficiency comprises selecting an effector protein with uniform enzyme activity across target loci with varying chromatin complexity, selecting an effector protein with enzyme activity limited to areas of open chromatin accessibility, preferably, wherein chromatin accessibility is measured using one or more of ATAC-seq, or a DNA-proximity ligation assay.   
     
     
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         105 . The method of  claim 95 , wherein selecting an optimized guide RNA comprises optimizing gRNA stability, gRNA immunogenicity, or both,
 preferably, wherein optimizing gRNA stability and/or gRNA immunogenicity comprises RNA modification,   preferably, wherein the modification comprises removing 1-3 nucleotides form the 3′ end of a target complementarity region of the gRNA; or wherein modification comprises an extended gRNA and/or trans RNA/DNA element that create stable structures in the gRNA that compete with gRNA base pairing at a target of off-target loci, or extended complimentary nucleotides between the gRNA and target sequence, or both.   
     
     
         106 . (canceled) 
     
     
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         108 . The method of  claim 11 , wherein the mode of delivery comprises delivering gRNA and/or CRISPR effector protein, delivering gRNA and/or CRISPR effector mRNA, or delivery gRNA and/or CRISPR effector as a DNA based expression system,
 preferably, wherein the mode of delivery further comprises selecting a delivery vehicle and/or expression systems from the group consisting of liposomes, lipid particles, nanoparticles, biolistics, or viral-based expression/delivery systems.   
     
     
         109 . (canceled) 
     
     
         110 . The method of  claim 108 , wherein expression is spatiotemporal expression is optimized by choice of conditional and/or inducible expression systems, including controllable CRISPR effector activity optionally a destabilized CRISPR effector and/or a split CRISPR effector, and/or cell- or tissue-specific expression system. 
     
     
         111 .- 182 . (canceled)