TJ Cradick
Cell & Gene Therapy Insights 9 (9), 1287–1292
November 8, 2023
An interview discussing the use of in vivo multiplex gene editing to target latent HIV, the evolution of the gene editing field, future applications, and other topics.
T Burdo, C Chen, R Kaminski, I K Sariyer, P Mancuso, M Donadoni, MD Smith, R Sariyer, M Caocci, S Liao1, H Liu, W Huo, H Zhao, J Misamore, MG. Lewis, V Simonyan, E Y. Xu, TJ Cradick, J Gordon & K Khalili
Gene Therapy (2023)
January 12, 2024
NHP demonstration of the safety and utility of CRISPR-Cas9 gene editing technology for in vivo editing of proviral DNA in ART-treated, virally controlled simian immunodeficiency virus (SIV) infected rhesus macaques, an established model for HIV infection.
EBT-001 is an AAV9-based vector delivering SaCas9 and dual guide RNAs designed to target multiple regions of the SIV genome: the viral LTRs, and the Gag gene.
A Cereseto,TJ Cradick & K Davies
The CRISPR Journal 4 (2), 166-168
April 20, 2021
New tweaks in the base editing toolbox signal offer promise in precision therapeutics for sickle-cell disease.
TJ Cradick
Chapter in Novel Immunotherapeutic Approaches to the Treatment of Cancer. Springer.
*Please request PDF file using Contact Us page.
May 31, 2016
Adoptive cell therapy using T cells that have been edited to express engineered chimeric antigen receptors (CARs) or T-cell receptors (TCRs) have succeeded in targeting patient tumors. Second and third generation CAR T cells have more modifications, including the addition of costimulatory molecules, and have shown improved results. A number of systems have been used for efficient knockin of the CAR or TCR and in the added gene editing steps, such as knocking out the endogenous TCR. As with therapeutic gene editing, improving delivery and reducing the possibility of off-target events are important criteria for optimizing ZFN, TALEN, and CRISPR/Cas nuclease editing.
PD Hsu, DA Scott, JA Weinstein, FA Ran, S Konermann, V Agarwala, Y Li, Eli J Fine, Xuebing Wu, Ophir Shalem, TJ Cradick, Luciano A Marraffini, Gang Bao, & Feng Zhang
July 21, 2013
Characterization of SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Evaluating >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci reveals that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification.
TJ Cradick, EJ Fine, CJ Antico, G Bao
Nucleic Acids Research 41 (20), 9584-9592
August 11, 2013
CRISPR/Cas9 systems need to be carefully designed to avoid potential off-target cleavage sites. Here we demonstrate that some CRISPR/Cas9 systems targeting the human hemoglobin β and C-C chemokine receptor type 5 genes have substantial off-target cleavage, especially within the hemoglobin δ and C-C chemokine receptor type 2 genes, respectively, causing gross chromosomal deletions.
The repair of the on-and off-target cleavage resulted in a wide variety of insertions, deletions, and point mutations that are plotted as an indel spectrum.
Y Lin, TJ Cradick, MT Brown, H Deshmukh, P Ranjan, N Sarode, BM Wile, PM Vertino, FJ Stewart, & G Bao
Nucleic Acids Research 42 (11), 7473-7485
May 16, 2014
A better understanding of the CRISPR/Cas9 specificity is needed to minimize off-target cleavage. Here we show that genomic sites could be cleaved by CRISPR/Cas9 systems when DNA sequences contain insertions (‘DNA bulge’) or deletions (‘RNA bulge’) compared to the RNA guide strand, and Cas9 nickases used for paired nicking can also tolerate bulges in one of the guide strands. Variants of single-guide RNAs (sgRNAs) for four endogenous loci were used as model systems, and their cleavage activities were quantified at different positions with 1- to 5-bp bulges. We further investigated 114 putative genomic off-tar get loci of 27 different sgRNAs and confirmed 15 off-target sites, each harboring a single-base bulge and one to three mismatches to the guide strand.
Our results strongly indicate the need to perform comprehensive off-target analysis related to DNA and sgRNA bulges in addition to base mismatches, and suggest specific guidelines for reducing potential off-target cleavage.
L Ye, J Wang, AI Beyer, F Teque, TJ Cradick, Z Qi, JC Chang, & G Bao
Proceedings of the National Academy of Sciences 111 (26), 9591-9596
March 26, 2014
In this study, we generated induced pluripotent stem cells (iPSCs) homozygous for the naturally occurring CCR5Δ32 mutation through genome editing of wild-type iPSCs using a combination of transcription activator-like effector nucleases (TALENs) or RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 together with the piggyBac technology.
TJ Cradick, P Qiu, CM Lee, EJ Fine, & G Bao
Molecular Therapy-Nucleic Acids 3, e214
December 2, 2014
COSMID (CRISPR Off-target Sites with Mismatches, Insertions, and Deletions) is a bioinformatics web tool that searches genomes for potential off-target sites (http://crispr.bme.gatech.edu). Based on the user-supplied guide strand and input parameters, COSMID identifies potential off-target sites with a specified number of mismatched bases and insertions or deletions when compared with the guide strand (bulges). For each site, amplification primers optimal for the chosen application are also given as output.
This ranked-list of potential off-target sites assists the choice and evaluation of intended target sites, thus helping the design of CRISPR/Cas systems with minimal off-target effects, as well as the identification and quantification of CRISPR/Cas-induced off-target cleavage in cells.
CL Ramirez, MT Certo, C Mussolino, MJ Goodwin, TJ Cradick, Anton P. McCaffrey, Toni Cathomen, Andrew M. Scharenberg, & J. Keith Joung
Nucleic Acids Research 40(12): 5560–5568.
Feb 28, 2012
The first publication on the nickase strategy, including a description of a simple and general method for converting engineered ZFNs into zinc finger nickases (ZFNickases) by inactivating the catalytic activity of one monomer in a ZFN dimer. ZFNickases show robust strand-specific nicking activity in vitro. In addition, we demonstrate that ZFNickases can stimulate HDR at their nicking site in human cells, albeit at a lower frequency than by the ZFNs from which they were derived. Finally, we find that ZFNickases appear to induce greatly reduced levels of mutagenic NHEJ at their target nicking site. ZFNickases thus provide a promising means for inducing HDR-mediated gene modifications while reducing unwanted mutagenesis caused by error-prone NHEJ. This ranked list of potential off-target sites assists the choice and evaluation of intended target sites, thus helping the design of CRISPR/Cas systems with minimal off-target effects, as well as the identification and quantification of CRISPR/Cas-induced off-target cleavage in cells.
TJ Cradick, K Keck, S Bradshaw, AC Jamieson, & AP McCaffrey
Molecular Therapy 18 (5), 947-954
February 16, 2010
Hepatitis B virus (HBV) chronically infects 350-400 million people worldwide and causes >1 million deaths yearly. Current therapies prevent new viral genome formation, but do not target pre-existing viral genomic DNA, thus curing only approximately 1/2 of patients. This manuscript described the first use of engineered nucleases to target a virus, using zinc-finger nucleases (ZFNs) to cleave HBV. Co-transfection of our ZFN pair with a target plasmid containing the HBV genome resulted in specific cleavage. Thirteen of 16 clones sequenced contained frameshift mutations that would lead to truncations of the viral core protein. These results demonstrate, for the first time, the possibility of targeting episomal viral DNA genomes using nucleases.
C Mussolino, J Alzubi, EJ Fine, R Morbitzer, TJ Cradick, T Lahaye, G Bao
Nucleic Acids Research 42 (10), 6762-6773
June 1, 2014
Engineered TALEN pairs targeting three human loci (CCR5, AAVS1 and IL2RG) were constructed. A detailed analysis of their activity, toxicity and specificity was performed. The TALENs showed comparable activity to benchmark ZFNs, with allelic gene disruption frequencies of 15–30% in human cells. Notably, TALEN expression was overall marked by a low cytotoxicity and the absence of cell cycle aberrations. Bioinformatics-based analysis of designer nuclease specificity confirmed partly substantial off-target activity of ZFNs targeting CCR5 and AAVS1 at six known and five novel sites, respectively. In contrast, only marginal off-target cleavage activity was detected at four out of 49 predicted off-target sites for CCR5- and AAVS1-specific TALENs. The rational design of a CCR5-specific TALEN pair decreased off-target activity at the closely related CCR2 locus considerably, consistent with fewer genomic rearrangements between the two loci. In conclusion, our results link nuclease-associated toxicity to off-target cleavage activity and corroborate TALENs as a highly specific platform for future clinical translation.
CM Lee, TJ Cradick, & G Bao
Molecular Therapy 24 (3), 645-654
January 19, 2016
We report the performance of the Neisseria meningitidis (Nme) CRISPR-Cas9 system that requires a longer protospacer-adjacent motif for site-specific cleavage, and present a comparison between the Spy and Nme CRISPR-Cas9 systems targeting the same protospacer sequence. The results with the native crRNA and tracrRNA, as well as a chimeric single guide RNA for the Nme CRISPR-Cas9 system were also compared. Our results suggest that, compared with the Spy system, the Nme CRISPR-Cas9 system has similar or lower on-target cleavage activity but a reduced overall off-target effect on a genomic level when sites containing three or fewer mismatches are considered. Thus, the Nme CRISPR-Cas9 system may represent a safer alternative for precision genome engineering applications.
M Müller, CM Lee, G Gasiunas, TH Davis, TJ Cradick, V Siksnys, & G Bao
Molecular Therapy 24 (3), 636-644
December 14, 2015
RNA-guided nucleases (RGNs) based on the type II CRISPR-Cas9 system of Streptococcus pyogenes (Sp) have been widely used for genome editing in experimental models. RGN specificity depends on both the guide RNA (gRNA) and the protospacer adjacent motif (PAM) recognized by the Cas9 protein. We generated RGNs based on two Streptococcus thermophilus (St) Cas9 proteins, which recognize longer PAMs, and performed a side-by-side comparison of the three RGN systems targeted to matching sites in two endogenous human loci, demonstrating that in samples with comparable on-target cleavage activities, significantly lower off-target mutagenesis was detected using St-based RGNs as compared to the standard Sp-RGNs. Moreover, similarly to SpCas9, the StCas9 proteins accepted truncated gRNAs, suggesting that the specificities of St-based RGNs can be further improved.
EJ Fine, TJ Cradick, CL Zhao, Y Lin, & G Bao
Nucleic Acids Research 42 (6), e42-e42
April 14, 2014
A description detailing the PROGNOS bioinformatic tool for ZFNs and TALENs, which also designs primers for a range of assays.
CM Lee, TJ Cradick, & G Bao
Molecular Therapy 24(3):475-87
February, 2016
The rapid advancement in targeted genome editing using engineered nucleases such as ZFNs, TALENs, and CRISPR/Cas9 systems has resulted in a suite of powerful methods that allows researchers to target any genomic locus of interest. A complementary set of design tools has been developed to aid researchers with nuclease design, target site selection, and experimental validation. Here, we review the various tools available for target selection in designing engineered nucleases, and for quantifying nuclease activity and specificity, including web-based search tools and experimental methods. We also elucidate challenges in target selection, especially in predicting off-target effects, and discuss future directions in precision genome editing and its applications.
Developing a Multiplex Gene Editing Approach Targeting Viral Infection
TJ Cradick
Cell & Gene Therapy Insights 9 (9), 1287–1292
November 8, 2023
An interview discussing the use of in vivo multiplex gene editing to target latent HIV, the evolution of the gene editing field, future applications, and other topics.
Preclinical Safety & Biodistribution of CRISPR Targeting SIV in Non-Human Primates
T Burdo, C Chen, R Kaminski, I K Sariyer, P Mancuso, M Donadoni, MD Smith, R Sariyer, M Caocci, S Liao1, H Liu, W Huo, H Zhao, J Misamore, MG. Lewis, V Simonyan, E Y. Xu, TJ Cradick, J Gordon & K Khalili
Gene Therapy (2023)
January 12, 2024
NHP demonstration of the safety and utility of CRISPR-Cas9 gene editing technology for in vivo editing of proviral DNA in ART-treated, virally controlled simian immunodeficiency virus (SIV) infected rhesus macaques, an established model for HIV infection.
EBT-001 is an AAV9-based vector delivering SaCas9 and dual guide RNAs designed to target multiple regions of the SIV genome: the viral LTRs, and the Gag gene.
Base Editors Flex Sights on Sickle-Cell Disease
A Cereseto,TJ Cradick & K Davies
The CRISPR Journal 4 (2), 166-168
April 20, 2021
New tweaks in the base editing toolbox signal offer promise in precision therapeutics for sickle-cell disease.
Cellular Therapies: Gene Editing & Next-Gen CAR T Cells
Chapter in Novel Immunotherapeutic Approaches to the Treatment of Cancer. Springer.
31 May 2016
Adoptive cell therapy using T cells that have been edited to express engineered chimeric antigen receptors (CARs) or T-cell receptors (TCRs) have succeeded in targeting patient tumors. Second and third generation CAR T cells have more modifications, including the addition of costimulatory molecules, and have shown improved results. A number of systems have been used for efficient knockin of the CAR or TCR and in the added gene editing steps, such as knocking out the endogenous TCR. As with therapeutic gene editing, improving delivery and reducing the possibility of off-target events are important criteria for optimizing ZFN, TALEN, and CRISPR/Cas nuclease editing.
DNA Targeting Specificity of RNA-guided Cas9 Nucleases
PD Hsu, DA Scott, JA Weinstein, FA Ran, S Konermann, V Agarwala, Y Li, Eli J Fine, Xuebing Wu, Ophir Shalem, J Cradick, Luciano A Marraffini, Gang Bao, & Feng Zhang
Nature Biotechnology 31 (9), 827-832
July 21, 2013
Characterization of SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Evaluating >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci reveals that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification.
CRISPR/Cas9 Systems Targeting β-Globin & CCR5 Genes Have Substantial Off-Target Activity
TJ Cradick, EJ Fine, CJ Antico, G Bao
Nucleic Acids Research 41 (20), 9584-9592
August 11, 2013
CRISPR/Cas9 systems need to be carefully designed to avoid potential off-target cleavage sites. Here we demonstrate that some CRISPR/Cas9 systems targeting the human hemoglobin β and C-C chemokine receptor type 5 genes have substantial off-target cleavage, especially within the hemoglobin δ and C-C chemokine receptor type 2 genes, respectively, causing gross chromosomal deletions.
The repair of the on-and off-target cleavage resulted in a wide variety of insertions, deletions, and point mutations that are plotted as an indel spectrum.
CRISPR/Cas9 Systems Have Off-Target Activity with Insertions or Deletions Between Target DNA & Guide RNA Sequences
Y Lin, TJ Cradick, MT Brown, H Deshmukh, P Ranjan, N Sarode, BM Wile, PM Vertino, FJ Stewart, & G Bao
Nucleic Acids Research 42 (11), 7473-7485
May 16, 2014
A better understanding of the CRISPR/Cas9 specificity is needed to minimize off-target cleavage. Here we show that genomic sites could be cleaved by CRISPR/Cas9 systems when DNA sequences contain insertions (‘DNA bulge’) or deletions (‘RNA bulge’) compared to the RNA guide strand, and Cas9 nickases used for paired nicking can also tolerate bulges in one of the guide strands. Variants of single-guide RNAs (sgRNAs) for four endogenous loci were used as model systems, and their cleavage activities were quantified at different positions with 1- to 5-bp bulges. We further investigated 114 putative genomic off-tar get loci of 27 different sgRNAs and confirmed 15 off-target sites, each harboring a single-base bulge and one to three mismatches to the guide strand.
Our results strongly indicate the need to perform comprehensive off-target analysis related to DNA and sgRNA bulges in addition to base mismatches, and suggest specific guidelines for reducing potential off-target cleavage.
Seamless Modification of Wild-Type Induced Pluripotent Stem Cells to the Natural CCR5Δ32 Mutation Confers Resistance to HIV Infection
L Ye, J Wang, AI Beyer, F Teque, TJ Cradick, Z Qi, JC Chang, & G Bao
Proceedings of the National Academy of Sciences 111 (26), 9591-9596
March 26, 2014
In this study, we generated induced pluripotent stem cells (iPSCs) homozygous for the naturally occurring CCR5Δ32 mutation through genome editing of wild-type iPSCs using a combination of transcription activator-like effector nucleases (TALENs) or RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 together with the piggyBac technology.
COSMID: A Web-Based Tool for Identifying & Validating CRISPR/Cas Off-Target Sites
TJ Cradick, P Qiu, CM Lee, EJ Fine, & G Bao
Molecular Therapy-Nucleic Acids 3, e214
December 2, 2014
COSMID (CRISPR Off-target Sites with Mismatches, Insertions, and Deletions) is a bioinformatics web tool that searches genomes for potential off-target sites (http://crispr.bme.gatech.edu). Based on the user-supplied guide strand and input parameters, COSMID identifies potential off-target sites with a specified number of mismatched bases and insertions or deletions when compared with the guide strand (bulges). For each site, amplification primers optimal for the chosen application are also given as output.
This ranked-list of potential off-target sites assists the choice and evaluation of intended target sites, thus helping the design of CRISPR/Cas systems with minimal off-target effects, as well as the identification and quantification of CRISPR/Cas-induced off-target cleavage in cells.
Seamless Modification of Wild-Type Induced Pluripotent Stem Cells to the Natural CCR5Δ32 Mutation Confers Resistance to HIV Infection
L Ye, J Wang, AI Beyer, F Teque, TJ Cradick, Z Qi, JC Chang, & G Bao
Proceedings of the National Academy of Sciences 111 (26), 9591-9596
March 26, 2014
In this study, we generated induced pluripotent stem cells (iPSCs) homozygous for the naturally occurring CCR5Δ32 mutation through genome editing of wild-type iPSCs using a combination of transcription activator-like effector nucleases (TALENs) or RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 together with the piggyBac technology.
Engineered Zinc Finger Nickases Induce Homology-Directed Repair with Reduced Mutagenic Effects
CL Ramirez, MT Certo, C Mussolino, MJ Goodwin, TJ Cradick, Anton P. McCaffrey, Toni Cathomen, Andrew M. Scharenberg, & J. Keith Joung
Nucleic Acids Research 40(12): 5560–5568.
Feb 28, 2012
The first publication on the nickase strategy, including a description of a simple and general method for converting engineered ZFNs into zinc finger nickases (ZFNickases) by inactivating the catalytic activity of one monomer in a ZFN dimer. ZFNickases show robust strand-specific nicking activity in vitro. In addition, we demonstrate that ZFNickases can stimulate HDR at their nicking site in human cells, albeit at a lower frequency than by the ZFNs from which they were derived. Finally, we find that ZFNickases appear to induce greatly reduced levels of mutagenic NHEJ at their target nicking site. ZFNickases thus provide a promising means for inducing HDR-mediated gene modifications while reducing unwanted mutagenesis caused by error-prone NHEJ. This ranked list of potential off-target sites assists the choice and evaluation of intended target sites, thus helping the design of CRISPR/Cas systems with minimal off-target effects, as well as the identification and quantification of CRISPR/Cas-induced off-target cleavage in cells.
Zinc-Finger Nucleases as a Novel Therapeutic Strategy for Targeting Hepatitis B Virus DNAs
TJ Cradick, K Keck, S Bradshaw, AC Jamieson, & AP McCaffrey
Molecular Therapy 18 (5), 947-954
February 16, 2010
Hepatitis B virus (HBV) chronically infects 350-400 million people worldwide and causes >1 million deaths yearly. Current therapies prevent new viral genome formation, but do not target pre-existing viral genomic DNA, thus curing only approximately 1/2 of patients. This manuscript described the first use of engineered nucleases to target a virus, using zinc-finger nucleases (ZFNs) to cleave HBV. Co-transfection of our ZFN pair with a target plasmid containing the HBV genome resulted in specific cleavage. Thirteen of 16 clones sequenced contained frameshift mutations that would lead to truncations of the viral core protein. These results demonstrate, for the first time, the possibility of targeting episomal viral DNA genomes using nucleases.
TALENs Facilitate Targeted Genome Editing in Human Cells with High Specificity & Low Cytotoxicity
C Mussolino, J Alzubi, EJ Fine, R Morbitzer, TJ Cradick, T Lahaye, & G Bao
Nucleic Acids Research 42 (10), 6762-6773
June 1, 2014
Engineered TALEN pairs targeting three human loci (CCR5, AAVS1 and IL2RG) were constructed. A detailed analysis of their activity, toxicity and specificity was performed. The TALENs showed comparable activity to benchmark ZFNs, with allelic gene disruption frequencies of 15–30% in human cells. Notably, TALEN expression was overall marked by a low cytotoxicity and the absence of cell cycle aberrations. Bioinformatics-based analysis of designer nuclease specificity confirmed partly substantial off-target activity of ZFNs targeting CCR5 and AAVS1 at six known and five novel sites, respectively. In contrast, only marginal off-target cleavage activity was detected at four out of 49 predicted off-target sites for CCR5- and AAVS1-specific TALENs. The rational design of a CCR5-specific TALEN pair decreased off-target activity at the closely related CCR2 locus considerably, consistent with fewer genomic rearrangements between the two loci. In conclusion, our results link nuclease-associated toxicity to off-target cleavage activity and corroborate TALENs as a highly specific platform for future clinical translation.
The Neisseria Meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells
CM Lee, TJ Cradick, & G Bao
Molecular Therapy 24 (3), 645-654
January 19, 2016
We report the performance of the Neisseria meningitidis (Nme) CRISPR-Cas9 system that requires a longer protospacer-adjacent motif for site-specific cleavage, and present a comparison between the Spy and Nme CRISPR-Cas9 systems targeting the same protospacer sequence. The results with the native crRNA and tracrRNA, as well as a chimeric single guide RNA for the Nme CRISPR-Cas9 system were also compared. Our results suggest that, compared with the Spy system, the Nme CRISPR-Cas9 system has similar or lower on-target cleavage activity but a reduced overall off-target effect on a genomic level when sites containing three or fewer mismatches are considered. Thus, the Nme CRISPR-Cas9 system may represent a safer alternative for precision genome engineering applications.
Streptococcus Thermophilus CRISPR-Cas9 Systems Enable Specific Editing of The Human Genome
M Müller, CM Lee, G Gasiunas, TH Davis, TJ Cradick, V Siksnys, & G Bao
Molecular Therapy 24 (3), 636-644
December 14, 2015
RNA-guided nucleases (RGNs) based on the type II CRISPR-Cas9 system of Streptococcus pyogenes (Sp) have been widely used for genome editing in experimental models. RGN specificity depends on both the guide RNA (gRNA) and the protospacer adjacent motif (PAM) recognized by the Cas9 protein. We generated RGNs based on two Streptococcus thermophilus (St) Cas9 proteins, which recognize longer PAMs, and performed a side-by-side comparison of the three RGN systems targeted to matching sites in two endogenous human loci, demonstrating that in samples with comparable on-target cleavage activities, significantly lower off-target mutagenesis was detected using St-based RGNs as compared to the standard Sp-RGNs. Moreover, similarly to SpCas9, the StCas9 proteins accepted truncated gRNAs, suggesting that the specificities of St-based RGNs can be further improved.
An Online Bioinformatics Tool Predicts Zinc Finger & TALE Nuclease Off-Target Cleavage
EJ Fine, TJ Cradick, CL Zhao, Y Lin, & G Bao
Nucleic Acids Research 42 (6), e42-e42
April 1, 2014
A description detailing the PROGNOS bioinformatic tool for ZFNs and TALENs, which also designs primers for a range of assays.
Nuclease Target Site Selection for Maximizing On-Target Activity & Minimizing Off-Target Effects in Genome Editing
CM Lee, TJ Cradick, EJ Fine, & G Bao
Molecular Therapy 24(3):475-87
January 11, 2016
The rapid advancement in targeted genome editing using engineered nucleases such as ZFNs, TALENs, and CRISPR/Cas9 systems has resulted in a suite of powerful methods that allows researchers to target any genomic locus of interest. A complementary set of design tools has been developed to aid researchers with nuclease design, target site selection, and experimental validation. Here, we review the various tools available for target selection in designing engineered nucleases, and for quantifying nuclease activity and specificity, including web-based search tools and experimental methods. We also elucidate challenges in target selection, especially in predicting off-target effects, and discuss future directions in precision genome editing and its applications.