AsCas12a

Cas ID

RESOURCES

  • Species Acidaminococcus sp. BV3L6link
  • Genome Assembly NZ_AWUR01000016.1link
  • Gene ID HMPREF1246_RS03730link
  • Protein ID WP_021736722.1link
  • UniProtKB U2UMQ6link
  • CDD TIGR04330link
  • Nucleotide Sequence FASTA 1
  • Amino Acid Sequence FASTA 1
  • crRNA (Ex) FASTA 1
  • sgRNA Sequence (Ex) FASTA 1
  • tracrRNA Sequence (Ex) FASTA 1
  • Direct Repeat (Native) FASTA 1

CLASSIFICATION

  • Cas ID 3.4.3
  • Nuclease Activity Target dsDNA (or ssDNA) + trans-ssDNA activity. Staggered cut.
  • Targeting Requirement 5' PAM
  • gRNA and Multiplexability crRNA
  • Class 2 subtype V-A
  • PAM or PFS TTTN

PROPERTIES

  • Protospacer Length 23 1
  • PAM TTTV 2
  • Protein Weight (KDa) 151.2 1
  • RNP Weight (KDa) 165.1 1
  • CDS Length (nt) 3921 1
  • Number Amino Acids 1307 1
  • crRNA_Length (nt) 43 1
  • sgRNA Length (nt) 43 1



DESCRIPTION

Summary

In 2015, AsCas12a (previously AsCpf1) was characterized as an RNA-guided endonuclease capable of efficient mammalian genome-editing activity 1 and has subsequently become a staple in the CRISPR toolbox. Recognizing a thymine-rich 5'-TTTV-3' PAM on the 5' side of the protospacer (on the non-target strand), AsCas12a utilizes a single catalytic site to cleave RNA-complementary double-stranded DNA (cis cleavage), as well as indiscriminately cleave single-stranded DNA (trans cleavage), 3 upon RNA-guided DNA binding. This has led to its use in rapid and highly sensitive nucleic acid detection 4 3 . AsCas12a and related orthologs LbCas12a and FnCas12a generate staggered cuts within the protospacer but distal to the PAM 1 . Because AsCas12a does not require tracrRNA and can process the CRISPR array into crRNAs internally, it is a good tool for multiplex editing 5 .

Applications

Administering AsCas12a complexed with a guide has biotechnological applications in healthcare, agriculture, and fundamental research. CRISPR-based treatments employing AsCas12a Ultra, an engineered variant of AsCas12a, are currently under investigation in human clinical trials 6 . Target-activated trans DNase activity has also been harnessed for detection of specific viral nucleic acid sequences 4 in human patient specimens 3 .

Additionally, by leveraging AsCas12a's ability to introduce targeted genetic disruptions, scientists have built successful platforms for genetic screening 7 , multiplex editing 8 , and model system creation 6 9 . AsCas12a, and related enzyme LbCas12a, have also been harnessed for agricultural purposes, improving plant immunity and nutritional content for staple crops like rice 10 . A more comprehensive listing of all agricultural Cas12a-based engineering can be found in other resources 11 , but several examples may be found in the experimental section of CasPEDIA.

Experimental Considerations

When using AsCas12a and related orthologs, such as LbCas12a, MbCas12a and FnCas12a, several experimental considerations should be taken into account.

Multiple software tools, such as CHOPCHOP 12 and CRISPOR 13 , exist for guide design 14 to help identify suitable target sites throughout the genomes of several model organisms. The T-rich PAM requirements of AsCas12a allow it to complement the PAM restriction to G-rich genomic sites in Cas9 editing 2 . The protospacer-adjacent motif (PAM) sequence (TTTV) of Cas12a lies 5' of the target site on the non-target strand, in contrast to Cas9, whose PAM lies 3' of the target site on the non-target strand. It is important to note that the individual "staggered" cuts reported in the original discovery paper 1 represent only a subset of the cuts made by AsCas12a in an RNA-complementary DNA target; in reality, AsCas12a forms an ~5-nt gap in the non-target strand and an ~2-nt gap in the target strand 15 .

AsCas12a's ability to process its own pre-crRNA facilitates multiplex applications, as multiple crRNAs can be synthesized in one transcript off of a single promoter. If the experimentalist's goal is to induce homology-directed repair (HDR), Cas12a may be a particularly efficient choice of CRISPR endonuclease 16 17 18 . It is hypothesized that the efficiency with which Cas12a induces HDR stems from its staggered cut and the speed with which it releases the PAM-distal cleavage product 16 .

Engineered versions of AsCas12a exist that broaden its PAM targeting 19 , improve its nuclease activity 6 , or improve its specificity 20 . These variants may be better suited for certain genome-engineering goals than WT AsCas12a. Because Cas12a homologs cleave both the non-target strand and target strand using a single active site 21 , these enzymes cannot be trivially engineered to form DNA nicks in the way that Cas9 can 22 . A variant of AsCas12a originally reported to act as a nickase (AsCas12a R1226A) 23 was later revealed to have double-stranded DNase activity that was dramatically slowed relative to WT AsCas12a 15 .

Search Constructs on Addgeneaddgene_link

Tool_Type Tool Is_Tool_Applicable Notes Citation
Delivery and Expression RNP Yes Established to work as WT and with variants with improved nuclease activity 6
Delivery and Expression Lenti Yes Combinatorial genetic screening 24
Delivery and Expression AAV No exceeds ideal packaging limits unless split into two AAV NaN
Delivery and Expression LNP Yes Cas12a Ultra delivered as RNP using LNPs 25
Delivery and Expression EDV Yes In principle, but not yet demonstrated NaN
Delivery and Expression Other_Delivery Cell penetrating peptide A5K-TAT peptide delivered in trans (10-20 fold stiochiometric excess) 26
Delivery and Expression Other_Delivery Cell penetrating peptide TAT-HA2 fusion peptide delivered in trans 27
Guide Design Guide_Design_Algorithm NaN CRISPOR; CHOPCHOP guide design algorithims 13 12
Guide Design Plasmid_Design NaN Can produce multiple gRNA on one promoter to facilitate easy targeting of multiple loci. 8

Application_Type Description Pharmaceutical_or_Product_Name NCT Responsible_Party Delivery_Mechanism In_Vivo_or_Ex_Vivo_Editing Citation_or_Publications
Human_Clinical_Trial Ex Vivo: EDIT-301 is a phase 1/2 clinical trial to treat Sickle Cell Disease and related Hemoglobinopathies by modifying hemopoietic stem cells EDIT-301 NCT: NCT04853576 Editas Medicine, Inc. RNP Nucleofection Ex Vivo NaN
Plant_Agriculture Staple crops: Rice 10 , Maize 10 , Soybean 28 NaN NaN NaN NaN NaN NaN
Plant_Agriculture Other: Tobacco 28 NaN NaN NaN NaN NaN NaN
Non_Human_Primate EDIT-201 is in preclinical development for NK cell therapy targeting solid tumors (No longer under development) EDIT-201 NaN Editas Medicine, Inc. RNP Nucleofection Ex vivo NaN

Tool_Type Tool_Name Description Citation
Diagnostic DNA endonuclease-targeted CRISPR trans reporter (DETECTR) Attomolar sensitivity DNA detection 3
Diagnostic Specific high-sensitivity enzymatic reporter unlocking (SHERLOCKv2) Attomolar sensitivity DNA detection 4

Variant_Name Description citations
enAsCas12a (E174R/S542R/K548R) Improved nuclease activity (indels) and specificity vs WT in mammalian cells20 20
AsCas12a Ultra (M537R/F870L) Dramatically improved nuclease activity (indels) in mammalian cells6 6
RVR AsCas12a (TATV) (S542R/K548V/N552R) and RR AsCas12a (TYCV) (S542R/K607R) AsCas12a variants capable of targeting with new PAM restrictions, TATV and TYCV, respectively19 19
AsCas12a Plus (E174R/R951K/R955A) Improved nuclease activity (indels) in mammalian cells29 29
KK AsCas12a (R951K, R955K) and KA (R951K,R955K) Precursor variants to AsCas12a Plus, KK and KA AsCas12a variants exhibit comparable on-target cleavage but lower activites with gRNA mismatches29 29

NUCLEOTIDE SEQUENCE


PROTEIN STRUCTURE

PFAM ID Description
PF18510 NUC
PF18501 REC1
PF18516 RuvC_1

Feature Type Start End Ligand Description Citations
Chain 1 1307 CRISPR-associated endonuclease Cas12a
DNA binding 599 607 PAM-binding on target DNA PubMed_ID:27114038;PubMed_ID:27444870
DNA binding 780 783 Target DNA PubMed_ID:27114038;PubMed_ID:27444870
DNA binding 951 968 Target DNA PubMed_ID:27114038;PubMed_ID:27444870
DNA binding 1051 1053 Target DNA PubMed_ID:27114038;PubMed_ID:27444870
Region 1 35 WED-I (OBD-I) PubMed_ID:27114038;PubMed_ID:27444870
Region 36 320 REC1 (helical-I) PubMed_ID:27114038;PubMed_ID:27444870
Region 321 526 WED-II (helical-II) PubMed_ID:27114038;PubMed_ID:27444870
Region 527 598 WED-II (OBD-I) PubMed_ID:27114038;PubMed_ID:27444870
Region 599 718 PI (LHD) PubMed_ID:27114038;PubMed_ID:27444870
Region 719 884 WED-III (OBD-III) PubMed_ID:27114038;PubMed_ID:27444870
Region 885 940 RuvC-I PubMed_ID:27114038;PubMed_ID:27444870
Region 941 957 Bridge helix PubMed_ID:27114038;PubMed_ID:27444870
Region 958 1066 RuvC-II PubMed_ID:27114038;PubMed_ID:27444870
Region 1067 1262 Nuclease domain PubMed_ID:27114038;PubMed_ID:27444870
Region 1263 1307 RuvC-III PubMed_ID:27114038;PubMed_ID:27444870
Coiled coil 74 106
Active site 800 800 For pre-crRNA processing
Active site 809 809 For pre-crRNA processing
Active site 860 860 For pre-crRNA processing
Active site 908 908 For DNase activity of RuvC domain PubMed_ID:27114038
Active site 993 993 For DNase activity of RuvC domain PubMed_ID:27114038
Active site 1226 1226 For DNase activity of nuclease domain PubMed_ID:27114038
Active site 1263 1263 For DNase activity of RuvC domain PubMed_ID:27114038
Binding site 47 51 crRNA (ChEBI:CHEBI:134528) PubMed_ID:27114038;PubMed_ID:27444870
Binding site 175 176 crRNA (ChEBI:CHEBI:134528) PubMed_ID:27114038;PubMed_ID:27444870
Binding site 307 310 crRNA (ChEBI:CHEBI:134528) PubMed_ID:27114038;PubMed_ID:27444870
Binding site 752 761 crRNA (ChEBI:CHEBI:134528) PubMed_ID:27114038;PubMed_ID:27444870
Binding site 806 808 crRNA (ChEBI:CHEBI:134528) PubMed_ID:27114038
Site 18 18 Binds crRNA PubMed_ID:27114038;PubMed_ID:27444870
Site 167 167 Binds PAM on target DNA PubMed_ID:27114038;PubMed_ID:27444870
Site 192 192 Binds crRNA PubMed_ID:27114038;PubMed_ID:27444870
Site 382 382 Binds crRNA-target DNA heteroduplex PubMed_ID:27114038;PubMed_ID:27444870
Site 548 548 Binds PAM on target DNA PubMed_ID:27114038;PubMed_ID:27444870
Site 607 607 Binds sequence-specific recognition of both target and non-target strand bases in PAM PubMed_ID:27114038;PubMed_ID:27444870
Helix 368 370 PDB_ID:5XH6
Site 872 872 Binds crRNA PubMed_ID:27114038;PubMed_ID:27444870
Site 1014 1014 Binds target DNA PubMed_ID:27114038
Mutagenesis 167 167 Wild-type to slightly improved guided indel formation. PubMed_ID:27114038
Mutagenesis 176 176 Decreased guided indel formation. PubMed_ID:27114038
Mutagenesis 192 192 Decreased guided indel formation. PubMed_ID:27114038
Mutagenesis 382 382 Nearly complete loss of guided indel formation. PubMed_ID:27114038
Mutagenesis 548 548 Decreased guided indel formation. PubMed_ID:27114038
Mutagenesis 604 604 Decreased guided indel formation. PubMed_ID:27114038
Mutagenesis 607 607 Nearly complete loss of guided indel formation, probable loss of PAM recognition. PubMed_ID:27114038
Mutagenesis 780 780 Nearly complete loss of guided indel formation. PubMed_ID:27114038
Mutagenesis 783 783 Complete loss of guided indel formation. PubMed_ID:27114038
Mutagenesis 908 908 Complete loss of guided indel formation; neither DNA strand is cleaved in vitro. PubMed_ID:27114038
Mutagenesis 951 951 Nearly complete loss of guided indel formation. PubMed_ID:27114038
Mutagenesis 955 955 Partial loss of guided indel formation. PubMed_ID:27114038
Mutagenesis 958 958 Partial loss of guided indel formation. PubMed_ID:27114038
Mutagenesis 993 993 Complete loss of guided indel formation; neither DNA strand is cleaved in vitro. PubMed_ID:27114038
Mutagenesis 1226 1226 Nearly complete loss of guided indel formation; nickase cleaves only the non-target DNA strand in vitro. PubMed_ID:27114038
Mutagenesis 1228 1228 Wild-type to slightly improved guided indel formation. PubMed_ID:27114038
Mutagenesis 1235 1235 About half loss of guided indel formation. PubMed_ID:27114038
Mutagenesis 1263 1263 Nearly complete loss of guided indel formation; neither DNA strand is cleaved in vitro. PubMed_ID:27114038
Helix 4 6 PDB_ID:5XH6
Beta strand 8 11 PDB_ID:5XH6
Beta strand 13 23 PDB_ID:5XH6
Helix 27 34 PDB_ID:5XH6
Helix 36 68 PDB_ID:5XH6
Helix 74 86 PDB_ID:5XH6
Helix 89 111 PDB_ID:5XH6
Beta strand 115 117 PDB_ID:5KK5
Helix 119 129 PDB_ID:5XH6
Turn 130 133 PDB_ID:5XH6
Helix 135 138 PDB_ID:5XH6
Helix 141 146 PDB_ID:5XH6
Helix 153 160 PDB_ID:5XH6
Turn 161 164 PDB_ID:5XH6
Helix 166 169 PDB_ID:5XH6
Helix 170 180 PDB_ID:5XH6
Helix 189 195 PDB_ID:5XH6
Helix 197 214 PDB_ID:5XH6
Helix 217 229 PDB_ID:5XH6
Helix 237 240 PDB_ID:5XH6
Helix 243 248 PDB_ID:5XH6
Beta strand 249 251 PDB_ID:5XH6
Helix 252 263 PDB_ID:5XH6
Helix 277 286 PDB_ID:5XH6
Helix 290 297 PDB_ID:5XH6
Helix 326 342 PDB_ID:5XH6
Helix 345 355 PDB_ID:5XH6
Turn 356 358 PDB_ID:5XH6
Helix 361 363 PDB_ID:5XH6
Helix 371 378 PDB_ID:5XH6
Beta strand 379 381 PDB_ID:5XH6
Helix 384 394 PDB_ID:5XH6
Helix 403 415 PDB_ID:5XH6
Helix 420 427 PDB_ID:5XH6
Helix 429 451 PDB_ID:5XH6
Helix 461 481 PDB_ID:5XH6
Helix 494 507 PDB_ID:5XH6
Helix 509 521 PDB_ID:5XH6
Beta strand 531 533 PDB_ID:5XH7
Turn 538 541 PDB_ID:5XH6
Helix 546 548 PDB_ID:5XH6
Helix 549 552 PDB_ID:5XH6
Beta strand 554 559 PDB_ID:5XH6
Beta strand 562 567 PDB_ID:5XH6
Beta strand 587 597 PDB_ID:5XH6
Helix 601 607 PDB_ID:5XH6
Turn 608 611 PDB_ID:5XH6
Helix 613 621 PDB_ID:5XH6
Beta strand 626 628 PDB_ID:5XH6
Beta strand 632 634 PDB_ID:5XH6
Beta strand 636 638 PDB_ID:5XH6
Helix 640 646 PDB_ID:5XH6
Beta strand 649 652 PDB_ID:5XH6
Beta strand 654 656 PDB_ID:5KK5
Helix 657 663 PDB_ID:5XH6
Helix 666 686 PDB_ID:5XH6
Turn 688 692 PDB_ID:5XH6
Helix 701 703 PDB_ID:5XH6
Helix 707 714 PDB_ID:5XH6
Helix 715 718 PDB_ID:5XH6
Beta strand 719 727 PDB_ID:5XH6
Helix 728 736 PDB_ID:5XH6
Beta strand 737 739 PDB_ID:5B43
Beta strand 741 746 PDB_ID:5XH6
Helix 748 750 PDB_ID:5XH6
Beta strand 751 753 PDB_ID:5KK5
Helix 760 768 PDB_ID:5XH6
Helix 771 775 PDB_ID:5XH6
Beta strand 778 781 PDB_ID:5XH6
Beta strand 786 790 PDB_ID:5XH6
Beta strand 804 807 PDB_ID:5XH6
Beta strand 812 817 PDB_ID:5B43
Helix 820 830 PDB_ID:5XH6
Helix 840 845 PDB_ID:5XH6
Helix 846 848 PDB_ID:5XH6
Beta strand 851 853 PDB_ID:5XH6
Helix 862 864 PDB_ID:5XH6
Beta strand 868 878 PDB_ID:5XH6
Beta strand 879 884 PDB_ID:5KK5
Helix 888 898 PDB_ID:5XH6
Beta strand 904 908 PDB_ID:5XH6
Beta strand 911 914 PDB_ID:5KK5
Beta strand 916 920 PDB_ID:5XH6
Beta strand 926 931 PDB_ID:5XH6
Beta strand 933 935 PDB_ID:5XH6
Helix 940 956 PDB_ID:5XH6
Helix 965 986 PDB_ID:5XH6
Beta strand 989 993 PDB_ID:5XH6
Helix 1011 1024 PDB_ID:5XH6
Beta strand 1038 1041 PDB_ID:5KK5
Beta strand 1057 1059 PDB_ID:5XH6
Beta strand 1062 1065 PDB_ID:5XH6
Beta strand 1070 1073 PDB_ID:5XH6
Turn 1075 1077 PDB_ID:5XH6
Helix 1085 1087 PDB_ID:5XH6
Helix 1091 1099 PDB_ID:5XH6
Beta strand 1101 1106 PDB_ID:5XH6
Turn 1108 1110 PDB_ID:5XH6
Beta strand 1113 1118 PDB_ID:5XH6
Helix 1123 1125 PDB_ID:5XH6
Beta strand 1134 1140 PDB_ID:5XH6
Beta strand 1145 1147 PDB_ID:5XH6
Beta strand 1153 1155 PDB_ID:5XH6
Beta strand 1159 1161 PDB_ID:5XH6
Beta strand 1174 1176 PDB_ID:5XH6
Helix 1178 1188 PDB_ID:5XH6
Helix 1200 1206 PDB_ID:5XH6
Helix 1209 1223 PDB_ID:5XH6
Beta strand 1226 1229 PDB_ID:5XH6
Turn 1230 1233 PDB_ID:5XH6
Beta strand 1234 1242 PDB_ID:5XH6
Beta strand 1248 1250 PDB_ID:5XH6
Helix 1251 1253 PDB_ID:5XH6
Helix 1262 1283 PDB_ID:5XH6
Beta strand 1284 1287 PDB_ID:5XH6
Helix 1295 1306 PDB_ID:5XH6


PDB ID: 5B43


  • 1. Zetsche, B., Gootenberg, J. S., Abudayyeh, O. O., Slaymaker, I. M., Makarova, K. S., Essletzbichler, P., Volz, S. E., Joung, J., van der Oost, J., Regev, A., Koonin, E. V., & Zhang, F. (2015). Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell, 163(3), 759–771. https://doi.org/10.1016/j.cell.2015.09.038 addgene_link
  • 2. Kim, H. K., Song, M., Lee, J., Menon, A. V., Jung, S., Kang, Y.-M., Choi, J. W., Woo, E., Koh, H. C., Nam, J.-W., & Kim, H. (2016). In vivo high-throughput profiling of CRISPR–Cpf1 activity. Nature Methods, 14(2), 153–159. https://doi.org/10.1038/nmeth.4104 addgene_link
  • 3. Chen, J. S., Ma, E., Harrington, L. B., Da Costa, M., Tian, X., Palefsky, J. M., & Doudna, J. A. (2018). CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science, 360(6387), 436–439. https://doi.org/10.1126/science.aar6245 addgene_link
  • 4. Gootenberg, J. S., Abudayyeh, O. O., Kellner, M. J., Joung, J., Collins, J. J., & Zhang, F. (2018). Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science, 360(6387), 439–444. https://doi.org/10.1126/science.aaq0179 addgene_link
  • 5. Zetsche, B., Heidenreich, M., Mohanraju, P., Fedorova, I., Kneppers, J., DeGennaro, E. M., Winblad, N., Choudhury, S. R., Abudayyeh, O. O., Gootenberg, J. S., Wu, W. Y., Scott, D. A., Severinov, K., van der Oost, J., & Zhang, F. (2016). Multiplex gene editing by CRISPR–Cpf1 using a single crRNA array. Nature Biotechnology, 35(1), 31–34. https://doi.org/10.1038/nbt.3737 addgene_link
  • 6. Zhang, L., Zuris, J. A., Viswanathan, R., Edelstein, J. N., Turk, R., Thommandru, B., Rube, H. T., Glenn, S. E., Collingwood, M. A., Bode, N. M., Beaudoin, S. F., Lele, S., Scott, S. N., Wasko, K. M., Sexton, S., Borges, C. M., Schubert, M. S., Kurgan, G. L., McNeill, M. S., … Vakulskas, C. A. (2021). AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines. Nature Communications, 12(1). https://doi.org/10.1038/s41467-021-24017-8
  • 7. DeWeirdt, P. C., Sanson, K. R., Sangree, A. K., Hegde, M., Hanna, R. E., Feeley, M. N., Griffith, A. L., Teng, T., Borys, S. M., Strand, C., Joung, J. K., Kleinstiver, B. P., Pan, X., Huang, A., & Doench, J. G. (2020). Optimization of AsCas12a for combinatorial genetic screens in human cells. Nature Biotechnology, 39(1), 94–104. https://doi.org/10.1038/s41587-020-0600-6 addgene_link
  • 8. Campa, C. C., Weisbach, N. R., Santinha, A. J., Incarnato, D., & Platt, R. J. (2019). Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts. Nature Methods, 16(9), 887–893. https://doi.org/10.1038/s41592-019-0508-6 addgene_link
  • 9. Lee, J. G., Ha, C. H., Yoon, B., Cheong, S.-A., Kim, G., Lee, D. J., Woo, D.-C., Kim, Y.-H., Nam, S.-Y., Lee, S., Sung, Y. H., & Baek, I.-J. (2019). Knockout rat models mimicking human atherosclerosis created by Cpf1-mediated gene targeting. Scientific Reports, 9(1). https://doi.org/10.1038/s41598-019-38732-2 addgene_link
  • 10. Malzahn, A. A., Tang, X., Lee, K., Ren, Q., Sretenovic, S., Zhang, Y., Chen, H., Kang, M., Bao, Y., Zheng, X., Deng, K., Zhang, T., Salcedo, V., Wang, K., Zhang, Y., & Qi, Y. (2019). Application of CRISPR-Cas12a temperature sensitivity for improved genome editing in rice, maize, and Arabidopsis. BMC Biology, 17(1). https://doi.org/10.1186/s12915-019-0629-5 addgene_link
  • 11. Bandyopadhyay, A., Kancharla, N., Javalkote, V. S., Dasgupta, S., & Brutnell, T. P. (2020). CRISPR-Cas12a (Cpf1): A Versatile Tool in the Plant Genome Editing Tool Box for Agricultural Advancement. Frontiers in Plant Science, 11. https://doi.org/10.3389/fpls.2020.584151
  • 12. Labun, K., Montague, T. G., Krause, M., Torres Cleuren, Y. N., Tjeldnes, H., & Valen, E. (2019). CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing. Nucleic Acids Research, 47(W1), W171–W174. https://doi.org/10.1093/nar/gkz365
  • 13. Concordet, J.-P., & Haeussler, M. (2018). CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Research, 46(W1), W242–W245. https://doi.org/10.1093/nar/gky354
  • 14. Hanna, R. E., & Doench, J. G. (2020). Design and analysis of CRISPR–Cas experiments. Nature Biotechnology, 38(7), 813–823. https://doi.org/10.1038/s41587-020-0490-7
  • 15. Cofsky, J. C., Karandur, D., Huang, C. J., Witte, I. P., Kuriyan, J., & Doudna, J. A. (2020). CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks. ELife, 9. CLOCKSS. https://doi.org/10.7554/elife.55143
  • 16. Singh, D., Mallon, J., Poddar, A., Wang, Y., Tippana, R., Yang, O., Bailey, S., & Ha, T. (2018). Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1 (Cas12a). Proceedings of the National Academy of Sciences, 115(21), 5444–5449. https://doi.org/10.1073/pnas.1718686115
  • 17. Hewes, A. M., Sansbury, B. M., & Kmiec, E. B. (2020). The Diversity of Genetic Outcomes from CRISPR/Cas Gene Editing is Regulated by the Length of the Symmetrical Donor DNA Template. Genes, 11(10), 1160. https://doi.org/10.3390/genes11101160
  • 18. Moreno-Mateos, M. A., Fernandez, J. P., Rouet, R., Vejnar, C. E., Lane, M. A., Mis, E., Khokha, M. K., Doudna, J. A., & Giraldez, A. J. (2017). CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing. Nature Communications, 8(1). https://doi.org/10.1038/s41467-017-01836-2 addgene_link
  • 19. Gao, L., Cox, D. B. T., Yan, W. X., Manteiga, J. C., Schneider, M. W., Yamano, T., Nishimasu, H., Nureki, O., Crosetto, N., & Zhang, F. (2017). Engineered Cpf1 variants with altered PAM specificities. Nature Biotechnology, 35(8), 789–792. https://doi.org/10.1038/nbt.3900 addgene_link
  • 20. Kleinstiver, B. P., Sousa, A. A., Walton, R. T., Tak, Y. E., Hsu, J. Y., Clement, K., Welch, M. M., Horng, J. E., Malagon-Lopez, J., Scarfò, I., Maus, M. V., Pinello, L., Aryee, M. J., & Joung, J. K. (2019). Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nature Biotechnology, 37(3), 276–282. https://doi.org/10.1038/s41587-018-0011-0 addgene_link
  • 21. Swarts, D. C., van der Oost, J., & Jinek, M. (2017). Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a. Molecular Cell, 66(2), 221-233.e4. https://doi.org/10.1016/j.molcel.2017.03.016 addgene_link
  • 22. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science, 337(6096), 816–821. https://doi.org/10.1126/science.1225829 addgene_link
  • 23. Yamano, T., Nishimasu, H., Zetsche, B., Hirano, H., Slaymaker, I. M., Li, Y., Fedorova, I., Nakane, T., Makarova, K. S., Koonin, E. V., Ishitani, R., Zhang, F., & Nureki, O. (2016). Crystal Structure of Cpf1 in Complex with Guide RNA and Target DNA. Cell, 165(4), 949–962. https://doi.org/10.1016/j.cell.2016.04.003
  • 24. Gier, R. A., Budinich, K. A., Evitt, N. H., Cao, Z., Freilich, E. S., Chen, Q., Qi, J., Lan, Y., Kohli, R. M., & Shi, J. (2020). High-performance CRISPR-Cas12a genome editing for combinatorial genetic screening. Nature Communications, 11(1). https://doi.org/10.1038/s41467-020-17209-1 addgene_link
  • 25. Suzuki, Y., Onuma, H., Sato, R., Sato, Y., Hashiba, A., Maeki, M., Tokeshi, M., Kayesh, M. E. H., Kohara, M., Tsukiyama-Kohara, K., & Harashima, H. (2021). Lipid nanoparticles loaded with ribonucleoprotein–oligonucleotide complexes synthesized using a microfluidic device exhibit robust genome editing and hepatitis B virus inhibition. Journal of Controlled Release, 330, 61–71. https://doi.org/10.1016/j.jconrel.2020.12.013
  • 26. Foss, D. V., Muldoon, J. J., Nguyen, D. N., Carr, D., Sahu, S. U., Hunsinger, J. M., Wyman, S. K., Krishnappa, N., Mendonsa, R., Schanzer, E. V., Shy, B. R., Vykunta, V. S., Allain, V., Li, Z., Marson, A., Eyquem, J., & Wilson, R. C. (2023). Peptide-mediated delivery of CRISPR enzymes for the efficient editing of primary human lymphocytes. Nature Biomedical Engineering, 7(5), 647–660. https://doi.org/10.1038/s41551-023-01032-2 addgene_link
  • 27. Zhang, Z., Baxter, A. E., Ren, D., Qin, K., Chen, Z., Collins, S. M., Huang, H., Komar, C. A., Bailer, P. F., Parker, J. B., Blobel, G. A., Kohli, R. M., Wherry, E. J., Berger, S. L., & Shi, J. (2023). Efficient engineering of human and mouse primary cells using peptide-assisted genome editing. Nature Biotechnology. https://doi.org/10.1038/s41587-023-01756-1 addgene_link
  • 28. Kim, H., Kim, S.-T., Ryu, J., Kang, B.-C., Kim, J.-S., & Kim, S.-G. (2017). CRISPR/Cpf1-mediated DNA-free plant genome editing. Nature Communications, 8(1). https://doi.org/10.1038/ncomms14406
  • 29. Huang, H., Huang, G., Tan, Z., Hu, Y., Shan, L., Zhou, J., Zhang, X., Ma, S., Lv, W., Huang, T., Liu, Y., Wang, D., Zhao, X., Lin, Y., & Rong, Z. (2022). Engineered Cas12a-Plus nuclease enables gene editing with enhanced activity and specificity. BMC Biology, 20(1). https://doi.org/10.1186/s12915-022-01296-1 addgene_link