Name: using sniper-cas9 to minimize off-target effects of crispr-cas9 without the loss of on-target activity via directed evolution
Uploaded: 2019-02-26T15:00:00
Duration: 11 min 37 s
Description: Watch this Scientific Journal Video about Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution at JoVE.com

This research was supported by grants from the Ministry of Science and ICT of Korea (2017M3A9B4061406) and the National Research Foundation of Korea (NRF) funded by the Korean government (MSIT) (grant numbers 2017M3A9B4061404 and 2018M3A9H3020844) to Jungjoon K. Lee. The plasmid encoding pGRG36 was a gift from Nancy Craig (Addgene plasmid #16666) and p11-LacY-wtx1 was a gift from Huimin Zhao.

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	<li>Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M., Joung, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+CRISPR-Cas+nuclease+specificity+using+truncated+guide+RNAs.">Improving CRISPR-Cas nuclease specificity using truncated guide RNAs.</a> Nature Biotechnology. 32 (3), 279-284 (2014).</li><li>Kim, S., Kim, D., Cho, S. W., Kim, J., Kim, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+RNA-guided+genome+editing+in+human+cells+via+delivery+of+purified+Cas9+ribonucleoproteins.">Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins.</a> Genome Research. 24 (6), 1012-1019 (2014).</li><li>Chen, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+proofreading+governs+CRISPR-Cas9+targeting+accuracy.">Enhanced proofreading governs CRISPR-Cas9 targeting accuracy.</a> Nature. 550 (7676), 407-410 (2017).</li><li>Kleinstiver, B. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-fidelity+CRISPR-Cas9+nucleases+with+no+detectable+genome-wide+off-target+effects.">High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.</a> Nature. 529 (7587), 490-495 (2016).</li><li>Slaymaker, I. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rationally+engineered+Cas9+nucleases+with+improved+specificity.">Rationally engineered Cas9 nucleases with improved specificity.</a> Science. 351 (6268), 84-88 (2016).</li><li>Casini, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+specific+SpCas9+variant+is+identified+by+in+vivo+screening+in+yeast.">A highly specific SpCas9 variant is identified by in vivo screening in yeast.</a> Nature Biotechnology. 36 (3), 265-271 (2018).</li><li>Lee, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution+of+CRISPR-Cas9+to+increase+its+specificity.">Directed evolution of CRISPR-Cas9 to increase its specificity.</a> Nature Communications. 9 (1), 3048 (2018).</li><li>Vakulskas, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-fidelity+Cas9+mutant+delivered+as+a+ribonucleoprotein+complex+enables+efficient+gene+editing+in+human+hematopoietic+stem+and+progenitor+cells.">A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells.</a> Nature Medicine. 24 (8), 1216-1224 (2018).</li><li>Anderson, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR+off-target+analysis+in+genetically+engineered+rats+and+mice.">CRISPR off-target analysis in genetically engineered rats and mice.</a> Nature Methods. 15, 512-514 (2018).</li><li>Kim, S., Bae, T., Hwang, J., Kim, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+high-specificity+Cas9+variants+using+sgRNAs+with+matched+5'+nucleotides.">Rescue of high-specificity Cas9 variants using sgRNAs with matched 5' nucleotides.</a> Genome Biology. 18 (1), 218 (2017).</li><li>Kulcsar, P. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crossing+enhanced+and+high+fidelity+SpCas9+nucleases+to+optimize+specificity+and+cleavage.">Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage.</a> Genome Biology. 18 (1), 190 (2017).</li><li>Zhang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfectly+matched+20-nucleotide+guide+RNA+sequences+enable+robust+genome+editing+using+high-fidelity+SpCas9+nucleases.">Perfectly matched 20-nucleotide guide RNA sequences enable robust genome editing using high-fidelity SpCas9 nucleases.</a> Genome Biology. 18 (1), 191 (2017).</li><li>McKenzie, G. J., Craig, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast,+easy+and+efficient:+site-specific+insertion+of+transgenes+into+enterobacterial+chromosomes+using+Tn7+without+need+for+selection+of+the+insertion+event.">Fast, easy and efficient: site-specific insertion of transgenes into enterobacterial chromosomes using Tn7 without need for selection of the insertion event.</a> BMC Microbiology. 6, 39 (2006).</li><li>Geissmann, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OpenCFU,+a+new+free+and+open-source+software+to+count+cell+colonies+and+other+circular+objects.">OpenCFU, a new free and open-source software to count cell colonies and other circular objects.</a> PLoS One. 8 (2), e54072 (2013).</li><li>Chen, Z., Zhao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+sensitive+selection+method+for+directed+evolution+of+homing+endonucleases.">A highly sensitive selection method for directed evolution of homing endonucleases.</a> Nucleic Acids Research. 33 (18), e154 (2005).</li><li>Kim, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR+RNAs+trigger+innate+immune+responses+in+human+cells.">CRISPR RNAs trigger innate immune responses in human cells.</a> Genome Research. 28 (3), 367-373 (2018).</li><li>Park, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cas-Analyzer:+an+online+tool+for+assessing+genome+editing+results+using+NGS+data.">Cas-Analyzer: an online tool for assessing genome editing results using NGS data.</a> Bioinformatics. 33, 286-288 (2017).</li><li>Studier, F. 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ToolGen has filed a patent application (PCT/KR2017/006212) covering Sniper-screen (status: pending, inventor: Jungjoon K. Lee). Jungjoon K. Lee, Joonsun Lee, Minhee Jung, and Euihwan Jeong are employees of ToolGen, Inc.

For those who want to avoid cumbersome screening procedures to obtain Sniper-Cas9, the Sniper-Cas9 protein and the encoding plasmid are available. Using these materials, the optimum length of the sgRNA, that providing the highest specificity ratio, should be determined. In addition, delivery of Sniper-Cas9 and sgRNA in an RNP format is recommended because it usually results in a higher specificity ratio than delivery in a plasmid format. Unlike Sniper-Cas9, other engineered Cas9 variants are not compatible with truncated sgRNAs6,7 or delivery in RNP form8 (with the exception of HiFi-Cas9).
For Sniper-screen, the selection of the mismatched sequence is the most important step. Selection of a mismatched sgRNA that is associated with low cleavage activity toward the GOI sequence should be avoided. If not, Cas9 variants with a WT level of specificity will not cleave the E. coli genomic DNA with the mismatched target site. This effect will result in a large number of background colonies, jeopardizing the whole screening procedure.
Because E. coli has a fast doubling time and high transformation efficiency compared to yeast, Sniper-screen is advantageous compared to yeast-based screening methods. Additionally, Sniper-screen should be more sensitive than other E. coli-based systems in which the mismatched site is carried on a plasmid: there is one copy of the genomic DNA and thus only one copy of the mismatched site in our system, but a large number of plasmids within a single E. coli cell.
The specificities of other DNA endonucleases that induce DSBs, such as SaCas9 or Cpf1s, could also be improved by using Sniper-screen. Unfortunately, Sniper-screen cannot be used to increase the specificity of base editors directly, because base editors do not induce DSBs in the genomic DNA of E. coli. As base editors use the nickase or dead version of Cas9 in the core of their system, the specificities of base editors could be increased by using the hits obtained from Sniper-screen.

In this paper, we aim to improve the specificity of Cas9 by combining different strategies. Various methods of avoiding the off-target effects of CRISPR-Cas9 have been developed. For example, truncated sgRNAs can be used to achieve higher specificity1. Additionally, the method of Cas9 delivery can be changed from a plasmid format to an RNP format to obtain higher specificity2. Specific amino acid residues of the Streptococcus pyogenes Cas9 (SpCas9) protein have been modified according to the rational design described previously3,4,5. Alternatively, amino acid residues have been altered in a random manner and the Cas9 variants with the highest specificity were identified using either a yeast6 or an E. coli7,8 screening system.
However, many groups have reported that Cas9 variants engineered using the design to debilitate the nonspecific interaction between Cas9 and the substrate exhibit low on-target activities7,8,9,10,11,12. We developed an E. coli-based directed evolution system, Sniper-screen, to screen randomly mutagenized Cas9 variants. An E. coli screening system has advantages over a yeast system because of the faster doubling time and higher transformation efficiencies of E. coli.
Both negative and positive selection, based on three different plasmids and a gene of interest (GOI) integrated into the E. coli genome, are used in Sniper-screen. Cas9 variants are expressed under the CMV-PltetO1 dual-promoter system of a low-copy number plasmid so that candidates identified in E. coli can be tested in mammalian cells without the need for subcloning. The GOI is introduced into the E. coli genome using the Tn7 transposon system. The sgRNA plasmid, which contains a temperature-sensitive origin of replication, expresses an sgRNA targeting the GOI; however, the sgRNA and GOI sequences are not perfectly matched. A perfectly matched sgRNA target site exists on a third plasmid containing the ccdB gene, which encodes a lethal product that poisons gyrase. In this system, cells expressing Cas9 variants with high off-target activities are removed because double-strand breaks (DSBs) are introduced into the mismatched site located in the genomic DNA. On the other hand, cells expressing Cas9 variants with low on-target activities are also removed because of lethal ccdB gene expression. The expression level of the Cas9 variants can be changed by altering the concentration of anhydrotetracycline (ATC), which adjusts the selection force.
We reasoned that locating the mismatched sgRNA target site in the genomic DNA rather than on a plasmid would increase the sensitivity of the system. The advantage of this approach is that there is only one genomic site, whereas there would be many plasmids, each containing a target site, within a single E. coli cell.
Using this system, we identified a Cas9 variant, Sniper-Cas9, which shows WT-level on-target activities and reduced off-target activities compared to WT Cas9. Sniper-Cas9 can achieve even higher specificity ratios by using truncated sgRNAs or RNP-based delivery rather than plasmid-based delivery.

<table>
	<tbody>
		<tr>
			<td>Alkaline Phosphatase, Calf Intestinal (CIP)</td>
			<td>NEB</td>
			<td>M0290L</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin Sodium Salt</td>
			<td>Fisher Chemical</td>
			<td>BP1760-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrotetracycline hydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>37919 or 94664</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic Antimycotic solution</td>
			<td>Welgene</td>
			<td>LS203-01</td>
			<td>Media component</td>
		</tr>
		<tr>
			<td>BamHI</td>
			<td>Enzynomics</td>
			<td>R003L</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Sigma Aldrich</td>
			<td>R4408</td>
			<td></td>
		</tr>
		<tr>
			<td>Diversify PCR Random Mutagenesis</td>
			<td>Clontech</td>
			<td>630703</td>
			<td>Error-prone PCR kit</td>
		</tr>
		<tr>
			<td>DNA-Shuffling Kit</td>
			<td>Jena Bioscience</td>
			<td>PP-103</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Welgene</td>
			<td>LM001-17</td>
			<td>Culture media</td>
		</tr>
		<tr>
			<td>Endura Electrocompetent Cells (DUO)</td>
			<td>Lucigen</td>
			<td>60242-2</td>
			<td>E. coli electrocompetent cells for library preparation</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Welgene</td>
			<td>S101-01</td>
			<td>Media component</td>
		</tr>
		<tr>
			<td>Gene Pulser II</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>E. coli electroporation equipment</td>
		</tr>
		<tr>
			<td>GeneMorph II Random Mutagenesis Kit</td>
			<td>Agilent</td>
			<td>200550</td>
			<td>Error-prone PCR kit</td>
		</tr>
		<tr>
			<td>genomic DNA prep kit</td>
			<td>GenAll</td>
			<td>106-152</td>
			<td>gDNA-prep kit</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>BioShop</td>
			<td>GLY001.1</td>
			<td></td>
		</tr>
		<tr>
			<td>GP/MP Cuvette, 0.1cm</td>
			<td>Bio-Rad</td>
			<td>BR165-2089</td>
			<td>E. coli electroporation equipment</td>
		</tr>
		<tr>
			<td>HEK293T cell</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td>Human cell line</td>
		</tr>
		<tr>
			<td>Kanamycin sulfate</td>
			<td>Acros Organics</td>
			<td>450810500</td>
			<td></td>
		</tr>
		<tr>
			<td>L-(+)-Arabinose</td>
			<td>Sigma Aldrich</td>
			<td>A3256-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>LaboPass PCR Purification Kit</td>
			<td>Cosmogenetech</td>
			<td>CMR0112</td>
			<td></td>
		</tr>
		<tr>
			<td>LaboPass Plasmid Mini</td>
			<td>Cosmogenetech</td>
			<td>CMP0112</td>
			<td>Mini-prep kit</td>
		</tr>
		<tr>
			<td>LB Agar Miller</td>
			<td>Formedium</td>
			<td>LMM0202</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Broth Miller</td>
			<td>Formedium</td>
			<td>LMM0102</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine</td>
			<td>Invitrogen</td>
			<td>11668019</td>
			<td>Transfection reagent</td>
		</tr>
		<tr>
			<td>NEBuilder HiFi DNA Assembly Master Mix</td>
			<td>NEB</td>
			<td>E2621L</td>
			<td>Gibson assembly (isothermal in vitro recombination) mixture</td>
		</tr>
		<tr>
			<td>Neon Transfection System</td>
			<td>Thermo</td>
			<td>MPK5000</td>
			<td>RNP Electroporation equipment</td>
		</tr>
		<tr>
			<td>Neon Transfection System 10 &#181;L Kit</td>
			<td>Thermo</td>
			<td>MPK1096</td>
			<td>RNP Electroporation equipment</td>
		</tr>
		<tr>
			<td>NucleoBond Xtra Midi EF</td>
			<td>Macherey-Nagel</td>
			<td>740420.50</td>
			<td>Midi-prep kit</td>
		</tr>
		<tr>
			<td>Oligo Clean &#38; Concentrator</td>
			<td>Zymo Research</td>
			<td>D4061</td>
			<td>DNA purification kit that enables low-volume elution</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Gibco</td>
			<td>LS31985070</td>
			<td>Transfection media</td>
		</tr>
		<tr>
			<td>p11-lacY-wtx1</td>
			<td>Addgene</td>
			<td>#123945</td>
			<td></td>
		</tr>
		<tr>
			<td>pgrg36</td>
			<td>Addgene</td>
			<td>#16666</td>
			<td>E. coli mutator strain</td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>NEB</td>
			<td>M0530L</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrophophatase</td>
			<td>NEB</td>
			<td>M2403L</td>
			<td></td>
		</tr>
		<tr>
			<td>Ribonucleotide Solution Set</td>
			<td>NEB</td>
			<td>N0450L</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase inhibitor</td>
			<td>NEB</td>
			<td>M0314L</td>
			<td></td>
		</tr>
		<tr>
			<td>T7 RNA polymerase</td>
			<td>NEB</td>
			<td>M0251L</td>
			<td></td>
		</tr>
		<tr>
			<td>Turbo Dnase</td>
			<td>Ambion</td>
			<td>AM2238</td>
			<td></td>
		</tr>
		<tr>
			<td>XbaI</td>
			<td>Enzynomics</td>
			<td>R013L</td>
			<td></td>
		</tr>
		<tr>
			<td>XL1-Red Competent Cells</td>
			<td>Agilent</td>
			<td>200129</td>
			<td></td>
		</tr>
	</tbody>
</table>

using sniper-cas9 to minimize off-target effects of crispr-cas9 without the loss of on-target activity via directed evolution

The development of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) into therapeutic modalities requires the avoidance of its potentially deleterious off-target effects. Several methods have been devised to reduce such effects. Here, we present an Escherichia coli-based directed evolution method called Sniper-screen to obtain a Cas9 variant with optimized specificity and retained on-target activity, called Sniper-Cas9. Using Sniper-screen, positive and negative selection can be performed simultaneously. The screen can also be repeated with other single-guide RNA (sgRNA) sequences to enrich for the true positive hits. By using the CMV-PltetO1 dual promoter to express Cas9 variants, the performance of the pooled library can be quickly checked in mammalian cells. Methods to increase the specificity of Sniper-Cas9 are also described. First, the use of truncated sgRNAs has previously been shown to increase Cas9 specificity. Unlike other engineered Cas9s, Sniper-Cas9 retains a wild-type (WT) level of on-target activity when combined with truncated sgRNAs. Second, the delivery of Sniper-Cas9 in a ribonucleoprotein (RNP) format instead of a plasmid format is possible without affecting its on-target activity.

After Sniper-screen is performed, the percentage of survival colonies can be calculated by dividing the number of colonies on the LB plate containing chloramphenicol, kanamycin, arabinose, and ATC (CKAA) by the number of colonies in the LB plate containing chloramphenicol and kanamycin only (CK). This percentage was usually very low when Sniper-screen was performed with the libraries of SpCas9. True-positive hits can be enriched by repeating the screen with the surviving pool. In this representative Sniper-screen, for example, a 100% survival rate was obtained after the third screen (Figure 1). Transfections using RNPs or plasmid-encoded Sniper-Cas9 can be done for various targets and the resulting on-target and off-target activities measured by targeted amplicon sequencing (Figure 2). At most targets, Sniper-Cas9 shows the same level of on-target activities and higher specificity ratios compared to the WT. Truncated sgRNAs can also be used to further improve specificity (Figure 3). However, their use is limited to only a few targets because they result in low on-target activities compared to full-length sgRNAs, in most cases. Therefore, sgRNAs with varying lengths (from 17- to 20-mers) must be tested and both on-target and off-target activities must be measured to optimize specificity.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59202/59202fig1.jpg" /> 
Figure 1: Representative Sniper-screens performed with different libraries of random Cas9 variants. Mutator library, EP library I, and EP library II indicate libraries made using different commercial kits. First, second, and final indicate the number of times the enrichment screen was performed7. This figure has been modified from Lee et al.7. <a href="https://www.jove.com/files/ftp_upload/59202/59202fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59202/59202fig2.jpg" /> 
Figure 2: On-target and off-target activities of WT-Cas9 or Sniper-Cas9 paired with a 20-mer guide sequence delivered via plasmid or RNP. Specificity ratios were determined by dividing the on-target activity by the off-target activity. Error bars indicate SEM (n = 3)7. This figure has been modified from Lee et al.7. <a href="https://www.jove.com/files/ftp_upload/59202/59202fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59202/59202fig3.jpg" /> 
Figure 3: On-target and off-target activities of Sniper-Cas9 compared to WT-Cas9 obtained using sgRNAs with variable lengths targeting the FANCF01 and AAVS sites. Specificity ratios were determined by dividing indel frequencies at on-target sites by those at the respective off-target sites. sgRNAs with a matched guanine at the 5&#39; terminus (GX18 or GX19) and those with a mismatched guanine (gX17, gX18, gX19, or gX20) are indicated. Specificity ratios were not calculated when the normalized on-target activities were &#60;70%7. This figure has been modified from Lee et al.7. <a href="https://www.jove.com/files/ftp_upload/59202/59202fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution at JoVE.com

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

Here, we present a protocol to optimize CRISPR-Cas9 to achieve a higher specificity without the loss of on-target activity. We use a directed evolution approach called Sniper-screen to find a mutant Cas9 with the desired characteristics. Sniper-Cas9 is compatible with truncated single-guide RNAs and delivery in a ribonucleoprotein format, well-known strategies for achieving higher specificities.

The development of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) into therapeutic modalities requires the ...

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Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of ...

Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System

Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System

Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas&#8217; disease mainly in Latin America. In order to identify a novel drug target ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and ...

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

Restriction endonuclease (REase) specificity engineering is extremely difficult. Here we describe a multistep protocol that helps to produce REase ...

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species ...