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Cas endonucleases

Recombinant CRISPR-associated (Cas) proteins are RNA-guided endonucleases suitable for ribonucleoprotein (RNP) complex formation with sequence specific guide RNA for subsequent gene targeting both in vitro and in living cells. [1-8]

There are a number of different Cas proteins available (e.g. Cas12a or CasX) [6-8] however, the most commonly used CRISPR/Cas system consists of Streptococcus pyogenes Cas9 (SpCas9) in combination with SpCas9-specific sgRNA.[1-5]

Delivery of Cas endonucleases as ribonucleoprotein (RNP) complex with sequence-specific guide RNA is highly efficient.[9-10] Delivery as Cas mRNA is an alternative approach in some cases (e.g. hard to transfect cells).

Cas endonucleaseOrganism isolated fromPAM sequence (5'-3')NLS positionFunctionality
SpCas9 (wt) 3NLSStreptococcus pyogenesNGG3x C-terminaldouble strand breaks (wildtype (wt))
SpCas9 (D10A) 3NLSStreptococcus pyogenesNGG3x C-terminalsingle-strand breaks/nicks (nickase),
lack of RuvC-like (D10A) nuclease activity
SpCas9 (D10A/H840A) 3NLSStreptococcus pyogenesNGG3x C-terminalno nuclease but DNA-binding activity (dead Cas9), lack of RuvC-like (D10A) and HNH-like (H840A) activity

Selected References

[1] Wang et al. (2016) CRISPR/Cas9 in Genome Editing and Beyond. Annu. Rev. Biochem. 85:227.
[2] Liu et al. (2019) CasX enzymes comprise a distinct family of RNA-guided genome editors. Nature 566:218.
[3] Gaj et al. (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31(7):397.
[4] Aldi et al. (2018) The CRISPR tool kit for genome editing and beyond. Nature Communications 9:1911.
[5] Pickar-Oliver et al. (2018) The next generation of CRISPR–Cas technologies and applications. Nature Reviews Molecular Cell Biology 20:507.
[6] Li et al. (2018) Design and assessment of engineered CRISPR–Cpf1 and its use for genome editing. Nature Protocols 13(5):899.
[7] Strecker et al. (2019) Engineering of CRISPR-Cas12b for human genome editing. Nature Communications 10:212.
[8] Liu et al. (2019) CasX enzymes comprise a distinct family of RNA-guided genome editors. Nature 566:218.
[9] DeWitt et al. (2017) Genome editing via delivery of Cas9 ribonucleoprotein. Methods 121:9.
[10] Kim et al. (2014) Highly efficient RNA-guided genome editing in humancells via delivery of purified Cas9 ribonucleoproteins. Genome Research 24:1012.