Engineered AsCas12a Variants with Enhanced Activity and Broadened PAM Compatibility

CRISPR-Cas12a (Cpf1) is a type V CRISPR effector RNA-guided DNA endonuclease with utility in synthetic biology and genome engineering. As an alternative to the commonly used Streptococcus pyogenes Cas9 (SpCas9), Cas12a recognizes TTTV (V = A/G/C) PAM sequences, which permits editing in AT-rich regio...

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Published in:Journal of biomolecular techniques 2019-12, Vol.30 (Suppl), p.S49-S49
Main Authors: Collingwood, Michael A, Vakulskas, Christopher A, Zhang, Liyang, Turk, Rolf, Bode, Nicole M, Jacobi, Ashley M, McNeill, Matthew S, McQuisten, Kyle, Thommandru, Bernice, Behlke, Mark A
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container_issue Suppl
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container_title Journal of biomolecular techniques
container_volume 30
creator Collingwood, Michael A
Vakulskas, Christopher A
Zhang, Liyang
Turk, Rolf
Bode, Nicole M
Jacobi, Ashley M
McNeill, Matthew S
McQuisten, Kyle
Thommandru, Bernice
Behlke, Mark A
description CRISPR-Cas12a (Cpf1) is a type V CRISPR effector RNA-guided DNA endonuclease with utility in synthetic biology and genome engineering. As an alternative to the commonly used Streptococcus pyogenes Cas9 (SpCas9), Cas12a recognizes TTTV (V = A/G/C) PAM sequences, which permits editing in AT-rich regions of the human genome. However, the extended length of AsCas12a PAM sequence relative to SpCas9 (TTTV vs. NGG) restricts its utility in genome editing. To improve the targeting range and enzymatic activity of Cas12a, we developed a bacterial-based selection assay to select for Cas12a mutants that demonstrate increased cleavage activity and reduced PAM specificity. We first selected Cas12a variants with enhanced activity at non-conical TTTT PAM, since this specific motif is significantly more prevalent throughout the human genome than the other three TTTV motifs. After multiple rounds of selections, we successfully enriched several variants from a Cas12a library with random mutations. We characterized the top enriched variants and revealed their enhanced cleavage activities at both TTTV and TTTT PAM sites when delivered into human cells as ribonucleoprotein (RNP) complexes. Combining mutations from these variants further enhanced cleavage activity, which globally enhanced the total genome editing efficiency of Cas12a over 96 sites found within several unique genomic loci. Early off-target site detection studies with GUIDE-Seq suggest that these mutants retain high on-target specificity of WT Cas12a with ribonucleoprotein (RNP)-based delivery. Overall, we anticipate that this new variant with enhanced activity and broadened PAM compatibility will allow for broader application of the CRISPR-Cas12a system for genome editing.
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title Engineered AsCas12a Variants with Enhanced Activity and Broadened PAM Compatibility
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