CRISPOR (citation) is a program that helps design, evaluate and clone guide sequences for the CRISPR/Cas9 system. CRISPOR Manual April 2021: MAD7, Thermocas9, SpRY, exons Full list of changes Step 1 Planning a lentiviral gene knockout screen? Use CRISPOR Batch Sequence name (optional): Enter a single genomic sequence, < 2300 bp, typically an exon Clear Box - Reset to default cttcctttgtccccaatctgggcgcgcgccggcgccccctggcggcctaaggactcggcgcgccggaagtggccagggcgggggcgacctcggctcacagcgcgcccggctattctcgcagctcaccatgGATGATGATATCGCCGCGCTCGTCGTCGACAACGGCTCCGGCATGTGCAAGGCCGGCTTCGCGGGCGACGATGCCCCCCGGGCCGTCTTCCCCTCCATCGTGGGGCGCC Text case is preserved, e.g. you can mark ATGs with lowercase. Instead of a sequence, you can paste a chromosome range, e.g. chr1:11,130,540-11,130,751 Step 2 Select a genome Homo sapiens - Human - UCSC Feb. 2009 (GRCh37/hg19) + SNPs: 1000Genomes, ExaC Note: pre-calculated exonic guides for this species are on the UCSC Genome Browser. We have 723 genomes, but not yours? Search NCBI assembly and send a GCF_/GCA_ ID to CRISPOR support. Step 3 Select a Protospacer Adjacent Motif (PAM) 20bp-NGG - Sp Cas9, SpCas9-HF1, eSpCas9 1.1 20bp-NNG - Cas9 S. canis 20bp-NGN - SpG 20bp-NNGT - Cas9 S. canis - high efficiency PAM, recommended 20bp-NAA - iSpyMacCas9 21bp-NNG(A/G)(A/G)T - Cas9 S. Aureus 20bp-NNG(A/G)(A/G)T - Cas9 S. Aureus with 20bp-guides 20bp-NG(G/T) - xCas9, recommended PAM, see notes 21bp-NNN(A/G)(A/G)T - KKH SaCas9 20bp-NNN(A/G)(A/G)T - KKH SaCas9 with 20bp-guides 20bp-NGA - Cas9 S. Pyogenes mutant VQR 24bp-NNNNCC - Nme2Cas9 20bp-NGCG - Cas9 S. Pyogenes mutant VRER 20bp-NNAGAA - Cas9 S. Thermophilus 20bp-NGGNG - Cas9 S. Thermophilus 20bp-NNNNG(A/C)TT - Cas9 N. Meningitidis 20bp-NNNNACA - Cas9 Campylobacter jejuni, original PAM 22bp-NNNNRYAC - Cas9 Campylobacter jejuni, revised PAM 22bp-NNNVRYAC - Cas9 Campylobacter jejuni, opt. efficiency 20bp-TTCN - CasX TTT(A/C/G)-23bp - Cas12a (Cpf1) - recommended, 23bp guides TTT(A/C/G)-21bp - Cas12a (Cpf1) - 21bp guides recommended by IDT TTTN-23bp - Cas12a (Cpf1) - low efficiency ATTN-23bp - BhCas12b v4 NGTN-23bp - ShCAST/AcCAST, Strecker et al, Science 2019 T(C/T)C(A/C/G)-23bp - TYCV As-Cpf1 K607R TAT(A/C/G)-23bp - TATV As-Cpf1 K548V TTTA-23bp - TTTA LbCpf1 TCTA-23bp - TCTA LbCpf1 TCCA-23bp - TCCA LbCpf1 CCCA-23bp - CCCA LbCpf1 GGTT-23bp - CCCA LbCpf1 YTTV-20bp - MAD7 Nuclease, Lui, Schiel, Maksimova et al, CRISPR J 2020 TTYN- or VTTV- or TRTV-23bp - enCas12a E174R/S542R/K548R - Kleinstiver et al Nat Biot 2019 20bp-NNNNCNAA - Thermo Cas9 - Walker et al, Metab Eng Comm 2020 20bp-NNN - SpRY, Walton et al Science 2020 20bp-NRN - SpRY (high efficiency PAM) 20bp-NYN - SpRY (low efficiency PAM) See notes on enzymes in the manual. Version 4.99 - Documentation - Contact us - Downloads/local installation - Citation - License